U.S. patent application number 14/982800 was filed with the patent office on 2016-07-28 for enzymatical modification of cell glycosylation using serum albumin and divalent cations.
The applicant listed for this patent is GLYKOS FINLAND LTD. Invention is credited to Heidi Anderson, Jari Natunen, Johanna Nystedt, Tero Satomaa, Leena Valmu.
Application Number | 20160215256 14/982800 |
Document ID | / |
Family ID | 40568564 |
Filed Date | 2016-07-28 |
United States Patent
Application |
20160215256 |
Kind Code |
A1 |
Nystedt; Johanna ; et
al. |
July 28, 2016 |
ENZYMATICAL MODIFICATION OF CELL GLYCOSYLATION USING SERUM ALBUMIN
AND DIVALENT CATIONS
Abstract
The invention is directed to a method and kit to control and
modify the status of cells, such as human stem cells, by changing
their glycosylation, in particular sialylation and fucosylation,
levels in a reaction condition where culture medium reagents, such
as divalent cations, are present and cells are kept non-adherent.
The invention is further directed to novel stem cells, the
glycosylation of which has been specifically altered.
Inventors: |
Nystedt; Johanna; (Helsinki,
FI) ; Anderson; Heidi; (Helsinki, FI) ; Valmu;
Leena; (Helsinki, FI) ; Natunen; Jari;
(Vantaa, FI) ; Satomaa; Tero; (Helsinki,
FI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLYKOS FINLAND LTD |
Helsinki |
|
FI |
|
|
Family ID: |
40568564 |
Appl. No.: |
14/982800 |
Filed: |
December 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13054541 |
Feb 15, 2011 |
9234169 |
|
|
PCT/FI2009/050628 |
Jul 16, 2009 |
|
|
|
14982800 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0006 20130101;
C12N 5/0663 20130101; A61K 2035/124 20130101; C12N 2501/724
20130101; C12N 5/0665 20130101; C12N 9/1051 20130101; C12P 21/005
20130101; C12N 2501/70 20130101 |
International
Class: |
C12N 5/00 20060101
C12N005/00; C12N 5/0775 20060101 C12N005/0775 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 2008 |
EP |
08397517.7 |
Claims
1.-41. (canceled)
42. A method for in vitro enzymatic modification of the
glycosylation of viable human adherent stem cells, the method
comprising the step of a) culturing human adherent stem cells in
non-NeuGc and serum free cell culture medium, wherein said human
adherent stem cells are from an aggregating or surface-adherent
cell culture, b) detaching said human adherent stem cells from a
cell culture surface, and c) contacting the human adherent stem
cells obtained in step b) in non-NeuGc and serum free culture
medium with a glycosyltransferase in conditions that prevent
adhesion of the human adherent stem cells, wherein adherence of the
human adherent stem cells is inhibited by providing shear force and
wherein said culture medium contains at least one divalent cation
Mg.sup.2+ or Ca.sup.2+ and wherein Mg.sup.2+ or Ca.sup.2+ ions are
used in a concentration of at least 0.05 mM.
43. The method according to claim 42, wherein the
glycosyltransferase is a fucosyltransferase or a
sialyltransferase.
44. The method according to claim 43, wherein the
fucosyltransferase is selected from the group consisting of FTIII,
FTIV, FTV, FTVI, FTVII and FTIX.
45. The method according to claim 43, wherein the
fucosyltransferase is a human fucosyltransferase.
46. The method according to claim 43, wherein the sialyltransferase
is selected from the group consisting of
.alpha.2,3-sialyltransferase and .alpha.2,6-sialyltransferase.
47. The method according to claim 43, wherein the sialyltransferase
is a human sialyltransferase.
48. The method according to claim 42, wherein the human adherent
stem cells are embryonal stem cells, cord blood stem cells,
hematopoietic stem cells, mesenchymal stem cells or human
peripheral blood mononuclear cells.
49. The method according to claim 42, wherein the culture medium
comprises a non-glycoprotein substantially without acceptor glycans
for glycosyltransferase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/054,541 filed Feb. 15, 2011, now U.S. Pat. No. 9,234,169,
issued Jan. 12, 2016, which is the U.S. national phase of PCT
Appln. No. PCT/FI2009/050628 filed Jul. 16, 2009 which claims
priority to European application. No. 08397517.7 filed Jul. 16,
2008, the disclosures of which are hereby incorporated in their
entirety by reference herein.
FIELD OF THE INVENTION
[0002] The invention is directed to a method and kit to control and
modify the status of cells, such as human stem cells, by changing
their glycosylation, in particular sialylation and fucosylation,
levels in a reaction condition where culture medium reagents, such
as divalent cations, are present and cells are kept non-adherent.
The invention is further directed to novel stem cells, the
glycosylation of which has been specifically altered.
BACKGROUND OF THE INVENTION
[0003] PCT/FI2006/050323 published 18.1.2007, describes a
modification of glycosylation of stem cells. The examples show
reaction of human cord blood mononuclear cells, comprising
hematopoietic stem cells, with sialyltransferase and
fucosyltransferases in MOPS buffer and 150 mM NaCl and by
sialidases in acetate buffer.
[0004] The "Sackstein" application (WO 2007/143204) indicates, and
their corresponding publication in Nature Medicine, filed after the
priority date of the above application, claims specifically
reaction conditions without any divalent cations and the
application specifically shows toxicity of MnCl.sub.2. Based on the
prior art it would not appear useful to use toxic Mn.sup.2+ or
other divalent cations Mg.sup.2+ or Ca.sup.2+-ions in the
reactions.
[0005] WO 2008/011094 also by Sackstein describes the use of
cytokines to stimulate glycosylation enzymes on hematopoietic stem
cells. The present invention is directed to the use of non-toxic
divalent cations, small molecules and supporting non-glycoprotein
albumin in context of in vitro modification of especially adherent
cell. It is realized that activation of cells by cytokines may also
produce undesired differentiation or cell activation leading to
negative activities in context of in vivo or ex vivo uses of the
cell.
[0006] PCT/FI2008/050015, filed Jan. 18, 2008, by the present
inventors indicates that the reactions even with the adherent cells
can be performed in the presence of Mg.sup.2+, which would be
preferred for the activity of modification enzymes, especially
sialyltransferase and fucosyltransferase. The example includes
reactions to mesenchymal stem cells in .alpha.-MEM, which contains
also other factors revealed useful by present invention. The
present invention revealed that use of both Mg.sup.2+ and Ca.sup.2+
ions preferably with supporting factors is especially useful for
producing optimally viable cells. The preferred conditions further
include conditions to prevent aggregation and surface binding of
cells, especially preferred shear force conditions.
[0007] PCT/FI2008/050015, filed Jan. 18, 2008, includes enzyme
tagging technologies. The present invention provides specific forms
and methods for the tagging, including glycan tagging with multiple
tags and limited lysine-specific tagging by biotinylation.
[0008] It is realised that previously published cell modification
conditions without divalent cations are not optimal for cell
status. It was revealed that the condition induced morphological
changes of cells, including non-natural granularity of the cells
which are likely to reduce the viability of the cells, though the
cells were indicated to be alive according to Sackstein et al.
[0009] The present invention is further directed to the use of
strong protease conditions producing unicellular cell suspension.
It is realized that this is needed to produce effective
glycosylation and optimal unicellular product for subsequent in
vivo use. It is realized that the cell population produced under
the Sackstein conditions with a low amount of protease might not
result in a unicellular preparation.
[0010] The process described by Sackstein and colleagues produces
an N-glycan-linked sialyl Lewis x (sLex) epitope, but essentially
no other selectin ligands structures (Sackstein et al. Nature Med
2008, 14(2):181-187). The present invention, including a reaction
condition with divalent cations, effectively produced the sLex on
O-glycan type structures different from the modification indicated
by Sackstein.
[0011] The present invention further provides methods for specific
tagging and washing cells with glycosyltransferase inhibitors to
remove the enzymes from cell preparations after modification
reactions.
[0012] Changes of sialylation by desialylation and resialylation
with specific sialyltransferases has been reported for red cells in
order to analyze binding specificities of influenza virus (Paulson,
J. et al.).
[0013] Partial desialylation and alpha-6-resialylation of human
peripheral blood and bone marrow CD34+ cells, both non-adherent
blood cells, has been reported, the peripheral blood cells having
been released by GM-CSF and most of the subjects being under cancer
therapy (Schwarz-Albiez, Reihard et al., 2004, Glycoconj. J.
21:451-459). The large variations in results may be due to therapy
and GM-CSF. The method used did not reveal quantitation of sialic
acid types, due to a limited specificity of the sialyltransferase
used. The modifications of sialic acid would likely further affect
the acceptor specificity of the sialyltransferase used and thus the
structures labelled. The present invention is especially directed
to .alpha.3-sialylation of the specific carrier structures.
[0014] Removal of NeuGc from pig xenotransplant tissue and
resialylation by NeuAc and sialyltransferase has been also
suggested (WO 2002/088351). That work was not directed to stem
cells, nor human stem cells directed methods, nor were the methods
used specified, although this is essential for applications in
these cells. The xenotransplantation idea is not relevant to
present invention due to tissue and species specificity of
glycosylation. A patent application (WO 2003/105908) describes
possible sialidase and sialyltransferase reactions for certain NK
or lymphocyte cell lines. The results revealed that the reactions
varied between the cell lines and were not predictable under the
conditions used in the work. Further, the reaction conditions of
sialyltransferase without CMP-sialic acid were not described by the
inventors.
[0015] Use of an inhibitor after glycomodification reaction has
been indicated, but the chemical nature of the inhibitors was not
indicated (WO 2004/072306). There is no indication of use of an
inhibitor, especially soluble acceptor which mimics competitive
inhibitor in context of cells and sialidase reactions or
glycosyltransferase reactions. Furthermore, no useful concentration
ranges for the substrates has been indicated and the present
inventors were first to reveal the need of removal of bound
glycosyltransferases from enzymes.
[0016] Xia et al. (2004) Blood 104 (10) 3091-9 describes changing
fucosylation of cord blood cell population without changing
sialylation levels and possible usefulness of the modification in
targeting of the cells to the bone marrow.
DIFFERENCE TO PRIOR ART
[0017] Manganese ion is most common divalent cation activator of
glycosyltransferase enzymes. Xia, L. et al (Blood 2004, 3091-96),
Hidalgo A. et al (Blood 2005, 567-575) and others have applied the
manganese method to hemtopoietic cells, which appears to be
somewhat tolerable to the cells during short time. However, the
detrimental effect to cells would partially reduce the usefulness
of the cells. Furthermore the manganese cannot be removed totally
from cell preparations and it is known to cause Parkinson's disease
like disorders with very low concentrations. It can be thus
concluded that the manganese methods create less viable or less
functional cells, which contain harmful or toxic metal
contamination, especially not desired for in vivo works.
[0018] The works of Sackstein (Nature Medicine 2008, 14(2):181-187
and WO2007/143204) indicate that it is possible to fucosylate cells
without presence of any divalent cations. The Sackstein refers to
obvious toxicity of manganese. Interestingly Sackstein selects
media devoid of also other divalent Mg2+ and Ca2+. This is likely
because these cations cause adherence, especially adherence to
surface and intracellular aggregation. The adherence blocks
sterically the cells from glycomodification. The present inventors
were able to prevent this by using methods preventing adherence of
cells and still obtain effective glycomodification.
[0019] The present inventors revealed glycomodification by enzymes
modifying N-acetyllactosamine sialylation (sialyltransferase and
sialidase) and fucosyltransferases, especially .alpha.3- and/or
.alpha.4-fucosyltransferase. It is realized that use of the
specific enzyme types under the complex reaction composition is
inventive. For example various divalent cations cause very enzyme
specifically activation or actual inhibition conditions of the
sialyltransfeases and .alpha.3/4-fucosyltransferase vary a lot, and
it would not have been easy to predict if the specific
enzymes/enzyme types would have been active enough to produce
effective cell fucosylation and sialylation modifications,
especially in the presence of two different cations.
[0020] Different enzyme types such as .alpha.2- or
.alpha.6-fucosyltransferases or ganglioseries ganglioside specific
sialyltrasnferases are not predicting the ion requirement or
concentrations useful for the preferred enzymes and in vitro
oligosaccharide conditions are not useful for prediction cell
surface reactions under complex conditions of the present
invention.
[0021] The transferases are under compex regulation in cells
including fosforylation and proteolysis may alter or destroy enzyme
activities, the present invention moved from traditional buffer
media depressing biological activities to complex biological
culture media including cations and cofactors activating cellular
enzymes and even chelating reagents among the vitamins, cofactors,
and even nucleotides known to inhibit glycosyltrasnferases and
amino acids. There are not useful models of predicting useful
reaction (if any) under such complex biological (close to in vivo)
conditions. In vivo the actual glycosyltransferase enzymes are
known to act for glycosylation of cells only under concentrated
intracellular Golgi membrane bound conditions, with specific
cofactors and ions but not as soluble extracellular enzymes.
[0022] The inventors were further able to perform the cellular
glycosylation in the presence of mammalian type glycosylated
proteins. It is realized that glycosyltransferases (or
glycosidases) can be inhibited or adhered with the relative large
amount of glycosyltransferases. The enzymes are known to bind
various glycans not even being their substrates or products
(product inhibition). The invention reveled glycoproteins
essentially devoid of glycan acceptors under the specific reaction
conditions including specifically controlled and limited acceptor
glycans. Most surprisingly the present invention reveals that it is
possible to produce glycomodification conditions where the cells
are modified in the presence of amount of glycoprotein comprising
limited and controlled actual acceptor(substrate) glycans for the
glycomodification enzymes. The limited and controlled glycan amount
can comprise the substrate glycan in excess, even large excess to
the cellular glycans and surpringly the invention revealed that
cells were effectively glycosylated. The presence of glycoproteins
is of benefit for supporting cells (e.g. transferrin), stabilizing
enzymes and further with effects preventing cell adherence.
[0023] The invention further revealed novel cell product with high
glycomodification levels, extremely high viability and presence of
normal morphology and absence of anomalous morphology of cells.
[0024] Novel Cell Products
[0025] The present invention further provides novel cell products
with improved glycomodification levels. The invention reveled
methods to produced extremely high amount of several sialyl-Lewis x
epitopes of very high portion of cells, preferably in over 40%,
more preferably over 60%, even more preferably over 70%, more
preferably over 80%, more preferably over 90%, even more preferably
over 95%, more preferably over 97%, and most preferably over 98% of
the modified cells when analyzed with specific antibodies of
present cells, in a preferred embodiment by CHO-131 directed to
O-glycans like ones on P-selectin ligands (different from
Sackstein's glycans).
[0026] The invention also reveled under sialyalted cells which
sialylation can be increased by present sialylation methods and
observed by specific reagents of invention. The preferred increased
proportion of cell surface sialylated cells, observable by the
specific reagent of examples, includes increased proportion of at
least 3%, more preferably at least 5%, even more preferably at
least 7% and most preferably at least 10% of the cells and under
specific embodiment 25% or more preferably 50% sialylation level
increase of the available cellular acceptor sites.
[0027] It is realized that the present methods allowed surprisingly
more efficient reaction by better enzyme activity and better
reaction kinetics with novel cell handling conditions. It is
further realized that the shear stress conditions applied remove
cell and extrracellular matrix residues from cell surfaces allowing
more effective glycomodification. Furthermore the present divalent
cation and cull culter media conditions allow recovery of cell
surface proteins and glycans after the stress from possible
transfer from cell culture conditions
[0028] The present invention further revealed optimized methods for
producing highly desialylated and viable stem cells by optimized
sialidase reactions.
[0029] The invention is further directed to combined sialylation
and fucosylation methods to allow production of increased amount,
of sialyl-Lewis x structures, the present optimized cell handling
methods allow thus production of the high sialyl-Lewis x expression
on high proportion of cells without producing high amounts of Lewis
x.
[0030] The novel cell products are further characterized by normal
morphology homogenously on very high proportion of the cells and
absence of anomalous morphology (e.g. ones produced with Sackstein
methods) and at least 98%, more preferably 99%, and most preferably
100% viability not observable with the cells produced by method of
Sackstein. It is realized that presence of anomalous or dead cells
even in minor amount can cause severe immunological complications
if cells are used in vivo.
[0031] The novel cell product does not contain substantial amounts
of manganese, because this is not used in the buffers. It can be
estimated that even after several cell washes manganese incubated
cells contain significant amount of toxic manganese. In present
cell preparation the manganese 2+ cation concentration is below 1
mM, more preferably below 100 microM, more preferably below 50
microM, even more preferably below 10 microM, even more preferably
below 1 microM, even more preferably below 100 nanoM, even more
preferably below 10 nanoM and even more preferably below 1 nanoM
and most preferably below 10 picoM.
[0032] Adherence
[0033] The present invention revelas that it is possible to prevent
adherence of cells in presence of non-toxic divalent cations in a
cell culture medium and that this allows effective
glycomodification of cells. The invention showed prevention of
adherence in form of adherence to surface and intracellular
aggregation.
[0034] It is realized that the methods can be applied to any
adherent cells such as Ca.sup.2+ and/or Mg.sup.2+ activatable
adherence receptor containing cells including integrin and C-type
(calsium activated) lectin, in preferred embodiment selectin such
as E-, P, or L-selectin, containing cells. Preferably such cells
includes epithelial, endothelial cell, cell growing as adherent
layers in cell culture, hematopoietic cells, leukocytes, especially
ones containing integrins, C-type lectins or selectins,
[0035] Glycomodification Reactions by Glycomodification Enzymes
including Glycosidases and Glycosyltransferases
[0036] The present invention is directed to glycomodification
reactions by glycosyltransferase enzymes referring herein to i)
preferred actual glycosyltranserase enzymes transferring
monosaccharide residues from nucleotide sugars to acceptors [more
specifically acceptor site(s) in acceptor glycans] and ii)
separately preferred transglycosylating enzymes which can
transglycosylate monosaccharide residues from non-nucleotide sugar
glycoconjugates such as from oligosaccharides, or monosaccharide
conjugates such as paranitrophenyl conjugates, or even free
monosaccharides iii) other glycan substituting enzymes,
transferring a non-monosaccharide substituent on to a glycan such
as sulphate residue transferred by a sulphotransferase.
[0037] The present invention provides novel conditions
glycomodification of cells especially glycomodification of N-linked
and O-linked glycans and optionally further N-acetyllactosamine
(Ga.beta.3/4GlcNAc)-epitopes of glycolipids. The N-acetyllactosamie
epitopes may be suitably sialyalted for a fucosylation reaction for
synthesis of sialylated and fucosylated epitopes
(.alpha.3-sialyalted to Gal, or .alpha.6-sialylated to GlcNAc on
type 1 N-acetyllactosamines) or sialylated for desialylation, and
the present invention further reveals sialation of
.alpha.3/4-fucosylated N-acetyllactosemines. The preferred
glycomodification is sialylation, desialylation, and/or
fucosylation of the N-acetyllactosamine epitopes. The preferred
sialylation is .alpha.3-sialylation of terminal Gal and/or
.alpha.6-sialylation (terminal Gal or the GlcNAc of
N-acetyllactosamine of cells, and fucosylation is .alpha.3- and/or
.alpha.4-fucosylation of GlcNAc residues of N-acetylalctosamine
structures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1. Surface glycan profiles of BM-MSC demonstrated by
FACS analysis of binding of plant lectins and glycoform-specific
antibodies after .alpha.2,3-sialylation (SAT),
.alpha.1,3-fucosylation (FUT), double SAT+FUT and sialidase (C.
perfringens) treatments.
[0039] BM-MSC were detached with 0.25% or 0.05% trypsin. The
control cells were analyzed directly after trypsinization. For the
enzyme reactions, 1.times.10.sup.6 cells were suspended in 300
.mu.L of .alpha.-MEM+0.5% human serum albumin, i.e., containing
divalent cations. The reactions were incubated for 1.5 or 2 hours
at +37.degree. C. and the reactions were resuspended every 20
minutes. The analyzed lectin populations are shown in the upper
panel.
[0040] FIG. 2. Microscopic visualization of BM-MSC in different
reaction buffer compositions (with and without divalent
cations).
[0041] 1.75.times.10.sup.6 cells were resuspended in either 300
.mu.l Minimum Essential Medium (MEM) a medium supplemented with
0.5% HSA or Ca.sup.2-/Mg.sup.2+-free Hanks' Balanced Salt Solution
(HBSS) supplemented with 0.1% HSA. The BM-MSC suspensions were
incubated in 24-well cell culture vessels for 2 hours in
+37.degree. C. cell incubator. A part of the reactions were
resuspended by mechanical pipetting every 20 minutes during the
incubation. Representative phase contrast microscope pictures of
cells in suspension were taken at the beginning of the experiment
and after 1 h and 2 h. Representative phase contrast microscope
pictures of each well have been taken with 10.times. objective of
only the cells in suspension. Panels A. and B. are enlarged
representative areas of 2 h situation where cells have been
resuspended every 20 minutes. Arrows indicate representative cells
with different morphology. Cells in .alpha.-MEM+0.5% HSA adhere
easily to small cell clusters if not resuspended at all during 2 h
incubation (encircled).
[0042] FIG. 3. Trypsinization inhibition (trypsin. stop) after 3
min by adding either excess .alpha.-MEM+10% fetal calf serum (FCS)
or .alpha.-MEM+10% human serum albumin (HSA).
[0043] Representative phase contrast microscope pictures of BM-MSC
whose trypsinizations were inhibited by either FCS (top) or HSA
(below). The cells were detached with either 0.25% or 0.05%
trypsin. Both trypsinization methods produced equally viable
unicellular cell suspensions and trypsinization could be as
effectively stopped with HSA as with FCS. Hence, xenoantigen-free
inhibition could be done.
[0044] FIG. 4. Phylogenetic tree of sialyltransferases.
[0045] FIG. 5. Removal of biotin-conjugated
.alpha.2,3-N-sialyltransferase (SAT) from solution by
streptavidin-coated magnetic particles, visualized by SDS-PAGE and
streptavidin-blotting.
[0046] Lanes 1-5, SAT without biotinylation; Lanes 6-9,
biotin-conjugated SAT. Lanes 1 and 6, magnetic particles with
precipitated SAT; Lanes 2-3 and 7-8, supernatant after the
precipitation; Lanes 4 and 9, supernatant of the subsequent wash
step; Lanes 5 and 10, SAT control (biotin-conjugated SAT visible at
the expected 39 kDa).
[0047] FIG. 6. Neutral N-glycan profile of bone-marrow derived
mesenchymal stem cells (BM-MSC) enzymatically modified with
.alpha.2,3-N-Sialyltransferase either when adherent with subsequent
EDTA (Versene) detachment or in suspension after EDTA (Versene)
detachment. Indicative terminal N-acetyllactosamine (LN) units for
successful sialylation are marked with red arrows.
[0048] FIG. 7. UCBMSC 391P (p4) in enforced suspension in cell
culture plate well (1) or conical tube (2). 0.5.times.10e6 cells
were used/300 .mu.l reaction buffer for 2 hours. Upper picture
shows MSCs in suspension at time point 0. Left: No mechanical
mixing (a) during 2 hour incubation aggregates cells (encircled)
and cells attach to plastic. Right: Mixing the cells every 30 min
(b) by pipetting significantly inhibits aggregation and cell
attachment. Cells are better maintained in one-cell suspensions
when kept in suspension in conical tubes (2b).
[0049] FIGS. 8A, 8B. Activity of .alpha.2,3 SAT in .alpha.-MEM with
human serum albumin (FIG. 8A) or without human serum albumin (FIG.
8B). Aliquots of reaction mixtures from overnight reaction were
analyzed by size-exclusion chromatography. Reaction product was
formed only in presence of albumin.
[0050] FIG. 9. Suspension incubated human umbilical cord
blood-derived mesenchymal stem cells (UCBMSC) in two different cell
densities. The cells were incubated in .alpha.MEM+0.5% human serum
albumin (HSA) for 2 hours with resuspension applied every 30 min.
Encircled areas indicate evident large cell aggregates.
[0051] FIGS. 10A, 10B. Human umbilical cord blood-derived
mesenchymal stem cell (UCBMSC) cell surface .alpha.2,3- and
.alpha.2,6-sialylation levels of freshly detached cells after
porcine trypsin-0.25% EDTA-based detachment (FIG. 10A) or TrypLE
Express detachment of the cells (FIG. 10B). Cell surface
.alpha.2,3- and .alpha.2,6-sialylation studied by labeling cells
with the conjugated plant lectins MAA, SNA and MAL-1 and flow
cytometry analysis. Percentage of gated positive cells indicated in
upper right corner.
[0052] FIGS. 11A-11D. Desialylation of 0.5.times.10e6 UCBMSCs using
200 mU Vibrio cholerae sialidase. MAA, SNA, MALI and CHO131
labeling of cells: FIG. 11A-2 h incubation control with reaction
buffer .alpha.MEM Glutamax +0.5% HSA and no enzyme; FIG. 11B-2 h
desialylation with 200 mU Vibrio cholerae sialidase in .alpha.MEM
Glutamax +0.5% HSA; FIG. 11C-2 h incubation control with reaction
buffer .alpha.MEM Glutamax +0.5% BSA and no enzyme; and FIG. 11D-2
h desialylation with 200 mU Vibrio cholerae sialidase in .alpha.MEM
Glutamax +0.5% BSA. Percentage positive gated cells indicated in
upper right corner. Results are presented as flow cytometry
scatters analyzed with FACSDiva software.
[0053] FIGS. 12A-12C. Acute (1 h) and subacute (12 h) in vivo
biodistribution of Tc99-HMPAO-(A,B) and [.sup.3H]-2DG (C)-labeled
human bone-marrow derived mesenchymal stem cells (BMMSC) with and
without enforced enzymatic cell surface .alpha.1,3-fucosylation
with FUTVI. The animals received 0.5.times.10.sup.6/mice (FIGS. 12A
and 12B) or 1.times.10.sup.6/mice (FIG. 12C) injected into the tail
vena. The radioactivity of whole lungs and the outflow of one femur
was counted. Control indicates animals receiving reaction buffer
incubated cells without enzyme and FUTVI indicates animals
receiving equal amounts of .alpha.1,3-fucosylated cells. The
results are presented either as means +SD or +SEM.
[0054] FIG. 13. Cells in 80% confluency after indicated
glycomodifications in culture media based reaction buffer
supplemented with 0.5% human serum albumin (HSA). All cells were
plated after the glycomodification with 1000 cell per cm.sup.2.
Both untreated cells and reaction buffer only incubated cells
served as controls. No differences were seen in growth behaviour or
morphology as compared to control cells.
[0055] FIG. 14. Acidic N-glycans of StemPro MSC SFM XF, 100.times.
Supplement.
DESCRIPTION OF THE INVENTION
[0056] The invention provides a novel method or kit for
modification of glycosylation of cells such as human stem cells.
The invention revealed that it is possible to modify glycosylation
of cells, when the modification is performed in presence of culture
medium (i.e. growth medium) reagents such as divalent cations
Mg.sup.2+ and/or Ca.sup.2+. The invention is especially directed to
glycosyltransferase modifications by sialyl- and
fucosyltransferases. Furthermore, the invention revealed specific
reagents for removal of the modification enzymes from reaction
mixtures including site specific tags of enzymes and specific
substrate inhibitors.
[0057] The invention is directed to a novel cell population and to
a method for its production. The cell population is derived from
human stem cells and the cell population comprises in vitro
enzymatically modified glycosylation produced by a method
comprising enzymatic in vitro glycan modification in the presence
of divalent cations Mg.sup.2- and Ca.sup.2+ and adherence of the
cells is inhibited by shear force. Optionally the modification
enzyme is removed from the cell preparation by using at least one
reagent selected from the group [0058] a glycan linked tag, not
essentially reducing the enzymatic activity of the protein; [0059]
a protein specifically linked tag, not essentially reducing the
enzymatic activity of the enzyme protein; [0060] a specific
substrate inhibitor binding effective to the active site of the
enzyme and releasing the enzyme from the cells, preferably being
acceptor substrate analog of glycosyltransferase or substrate
analog for protease.
[0061] The novel cells are preferably prepared or derived from cord
blood or bone marrow derived mesenchymal stem cells or other
mesenchymal stem cells. In a preferred embodiment the cells are
prepared or derived from isolated cord blood cells and in another
embodiment from bone marrow derived cells. The invention is
specifically directed to adherent cells. It is realized that
several modifications can change also other cell types adherent. In
a specific embodiment the invention is directed to modification of
stem cells by the methods according to the present invention, when
the adhesion of the cells is increased.
[0062] The novel cell population is preferably produced by a
method, wherein the cells are modified in the presence of
Mg.sup.2+. It was realized that Mg.sup.2+ would support the
glycosylation reactions. In another preferred embodiment the novel
cell population is preferably produced by method, wherein the cells
are modified in the presence of Ca.sup.2-. The invention revealed
that presence of Ca.sup.2+ is useful for maintaining the cell
morphology. Even more preferably the cells are modified in the
presence of both Ca.sup.2+ and Mg.sup.2+.
[0063] The invention is further directed to cell population and its
production, wherein the cells are modified in the presence of
additional supporting factors required for cell cultivation
selected from the group consisting of vitamins, energy and
structural nutrients, and physiological salt, i.e. in culture
medium such as .alpha.-MEM (see Table 12).
[0064] In another preferred embodiment the novel cells are modified
in the presence of non-glycoprotein.
[0065] In another preferred embodiment the cells are modified in
the presence of at least one cell supporting materials, preferably
all additional supporting factors selected from the group
consisting of
[0066] 1) Ca.sup.2+ and Mg.sup.2+;
[0067] 2) Supporting factors
[0068] 2.1) vitamins,
[0069] 2.2) energy and structural nutrients such as amino acids,
and
[0070] 2.3) physiological salt;
[0071] 3) non-glycoprotein.
[0072] The invention is specially directed to the combination of
the factors, which is especially useful for maintaining the cells
during the modification reactions.
[0073] In a preferred embodiment both Mg.sup.2- and Ca.sup.2+ ions
are used in a concentration of at least 0.05 mM, even more
preferably 0.5 mM and most preferably at least 1 mM and less than
10 mM. The preferred concentrations of the non-toxic divalent
cations are close to physiological concentration, preferably
between about 0.5 mM and 1.0 mM, more preferably about 0.8 mM for
Ca.sup.2-, and preferred Mg.sup.2+ concentration is between about
1.5 and 2 mM, more preferably about 1.8 mM. More preferably the
ions and optionally additional ions are used in concentrations
similar to those in the .alpha.-MEM medium, also referred to as the
minimal essential medium (Nature 1971, Catalog of
Gibco/Invitrogen.RTM.).
[0074] Accordingly, the kit of the present invention comprises
culture medium for cell cultures as defined above and
glycosyltransferase or glycosidase. The kit may further include
albumin, such as human albumin, or transferrin as a
non-glycoprotein or the culture medium may also contain said
non-glycoprotein as a supplement.
[0075] The preferred cell modification kits contain cell culture
medium (or its concentrate), enzyme and preferably also possible
cofactors of enzyme such as nucleotide sugar or other donor
substrates for the glycomodification enzyme. In a preferred
embodiment the enzyme, medium and nucleotides are in separate
containers, in another preferred embodiment the glycomodification
enzyme is included in the container of the medium and the cofactor
is in the separate container. In one preferred kit, all components
are in the same container in solid form. The preferred kit further
includes instructions for performing reaction with the required
components for suitable time and optionally instructions and/device
for mixing or suspending the cells.
[0076] Detaching Adherent Stem Cells by Protease Treatment
[0077] The invention is especially directed to novel cell
populations produced according to the invention, wherein the cells
are adherent cells detached from cell culture surface by protease
treatment. The cells are preferably detached under condition
producing unicellular cell suspension.
[0078] The preferred trypsin condition includes trypsin
concentrations from about 0.01 mg/ml to about 0.5 mg/ml, wherein 1
mg corresponds to about 10 000 BASE units of trypsin, as defined by
the producer of the trypsin (Invitrogen). The trypsin is preferably
from bovine (product number 25200, Invitrogen). The reaction is
preferably short, i.e. from about 0.5 min to about 5 min, more
preferably about 3 min.
[0079] In a preferred embodiment the cell population is produced
from strongly adherent cells and protease is trypsin which is used
in a condition equivalent of trypsin concentration of 0.075 mg/ml,
more preferably 1 mg/ml, most preferably 0.25 mg/ml for about three
minutes.
[0080] Cells Produced by Optimal Proteolytic Reaction.
[0081] The invention is especially directed to mesenchymal cells,
which are released from adherent cell culture by a protease,
preferably trypsin under optimal conditions (Example 3).
[0082] Production of Unicellular Cell Preparation.
[0083] The invention is directed to the release of adherent
mesenchymal stem cells from cell culture support under proteolysis
condition producing unicellular cell preparation. The invention
further revealed that the unicellular cell suspension is
effectively modified in glycomodification reactions (Examples 2 and
3).
[0084] As a control experiment, adherent cells which are not in
unicellular forms are not effectively modified by
glycosyltransferases (Example 9).
[0085] Use of Protease Inhibitors
[0086] The invention revealed that it is useful to end the protease
reaction by inhibitor molecules. In a preferred embodiment the
protease reaction is ended by adding an inhibitor of protease,
preferably substrate inhibitor which releases the protease from the
cell surface (Example 3).
[0087] The invention is further directed to the novel cells and
their production method, wherein the protease is effectively
removed from cell surface of the modified cells, preferably using a
covalent tag attached to the protease and/or a substrate inhibitor
of the protease, which is optionally tagged.
[0088] The protease enzyme and/or its substrate inhibitor are
preferably tagged by at least one covalent tagging method selected
from the group consisting of
[0089] protein amine tagging,
[0090] glycan tagging,
[0091] N-terminal tagging of protein or peptide and
[0092] C-terminal tagging of protein or peptide.
[0093] The invention is further directed to a method for removal of
the protease and/or protease inhibitor, wherein the protease and/or
substrate inhibitor of the protease is tagged and the inhibitor and
protease are effectively removed from the surface of the modified
cells by using an adsorbent binding the tag (Example 5).
[0094] In a preferred embodiment the protease is tagged and
high-affinity inhibitor peptide or protein is used.
[0095] Glycomodification of Cells in Presence of Non-Toxic Divalent
Cations and other Supporting Actors
[0096] The invention revealed that it is possible to modify
adherent cells, preferably adherent stem cells and most preferably
mesenchymal stem cells in presence of divalent cations, when the
divalent cation is non-toxic. The non-toxic divalent cation is
preferably not toxic as high micromolar or millimomolar
concentration. An example of a toxic divalent cation is Mn.sup.2+
cation. The present method is in the contrast to the method of
Sackstein wherein all divalent cations were omitted.
[0097] In a preferred embodiment the preferred non-glycoproteins of
present invention do not include cytokines according to WO
2008/011094.
[0098] The present invention reveals that in the presence of
specific divalent cations mesenchymal cells are more viable and can
be effectively glycosylated by glycosyltransferases, especially by
sialyltransferases and fucosyltransferases.
[0099] It was realized that the reaction media lacking divalent
cations is not optimal for mesenchymal stem cells. Though the cells
appear viable their ability to recover from the stress due to
proteolytic treatment is reduced and the cells tend to adhere to
aggregates which reduce both their viability and their glycans
accessibility to be modified. The invention revealed that
aggregation and surface attachment occurs in the presence of
non-toxic divalent cations. However, as shown in Example 2, it is
possible to recover the adherent mesenchymal stem cells. The
presence of the divalent cations provided 100% viability. This is a
clear benefit as the dead cells may cause undesired immunological
and other reactions in therapeutic use. Furthermore it was revealed
that resuspension of the cells prevented aggregation and surface
adhesion, hence, leading to a clearly increased yield of the cells
(Example 2).
[0100] The conditions in the Sackstein-process without divalent
cations can be regarded as non optimal to the activity for many
fucosyltransferases and sialyltransferases. The presence of
divalent cations is especially preferred for fucosyltransferase
reactions. Presence of divalent cations is especially preferred for
fucosyltransferases FUC-TIII-VII, more preferably for Fuc-TVI and
Fuc-TVII. Sialyltransferases are also known to function optimally
with divalent cations.
[0101] In a preferred embodiment both Mg.sup.2- and Ca.sup.2+ ions
are used with at least 0.05 mM, more preferably at least 0.1 mM,
even more preferably 0.5 mM and most preferably at least 1 mM and
less than 10 mM. More preferably the ions are use as physiological
concentrations, preferably similar as in .alpha.-MEM.
[0102] Mesenchymal Stem Cells in Suspension.
[0103] The invention revealed that the novel cells produced by the
present invention are maintained in mononuclear cell-like
morphology while the conditions without non-toxic divalent cations
caused granularity of the cells. The invention is especially
directed to the novel cell population, wherein the cells in
suspension are essentially mononuclear cell-like cells without
granularity (Example 2).
[0104] Glycomodification of Cells in Presence of Non-Glycoprotein
and Supporting Factors: Non-Glycoprotein
[0105] The invention is directed to glycomodification of adherent
cells, more preferably mesenchymal stem cells, in the presence of
non-glycoproteins including non-glycosylated or non-glycosylable
protein. The non-glycoprotein is a protein, which does not contain
substantial amount of acceptor glycans for modification enzymes,
such as terminal Gal, lactosamine(s), GalNAc, or subterminal
GlcNAc. The acceptor glycan means a substrate for
glycosyltransferases according to the present invention, preferably
.alpha.3- and/or .alpha.6-sialyltransferase acceptor sites and
substrates for glycosidases such as sialidase enzymes such as
.alpha.3- and/or .alpha.6-sialylglycans.
[0106] Preferred non-glycoproteins are protein capable of
supporting cell culture without preventing or substantially not
preventing the glycomodification reactions of the invention. In a
preferred embodiment the non-glycoprotein increases the effect of
glycosyltransferase reaction. This is surprising because the
proteins do not have obvious interactions. The invention showed
that albumin support glycosyltransferase reactions to cells and
that human albumin preparations have higher such activity when
compared to bovine serum albumin. The preferred non-glycoproteins
include preferably human non-glycoproteins, more preferably human
serum proteins, preferably non-glycoprotein forms of albumin and/or
transferrin, most preferably human albumin and human transferrin or
derivatives or fragments, preferably functional fragments
thereof.
[0107] Human serum proteins are for instance (see Quality assurance
and reproducibility Am. J. Clin. Path. 97: 97, 1992): 1)
transthyretin ("retinol binding protein"; "prealbumin"); 2)
albumin; 3) .alpha.-1 globulins, such as .alpha.-1 protease
inhibitor (.alpha.-1 antitrypsin), .alpha.-1 glycoprotein
(orosomucoid), .alpha.-fetoprotein and high density lipoprotein
(HDL); 4) .alpha.-2 globulins, such as .alpha.-2 macroglobulin,
antithrombin III, ceruloplasmin, and haptoglobin; 5) beta
globulins, such as beta and pre-beta lipoproteins (LDL and VLDL),
C3, C-reactive protein, haemoglobin, plasminogen, and transferrin
("principal component of the betal subdivision"); 6) gamma
globulins.
[0108] There are also additional human serum proteins and at least
325 distinct proteins have been identified (Pieper et al. 2003,
Proteomics 2003, 3:1345-1364).
[0109] The preferred non-glycoproteins include non-glycosylated
proteins such as serum albumin, which does not contain
glycosylations sites, in a preferred embodiment bovine serum
albumin and non-glycosylated isoforms of human serum albumin or
essentially non-glycoprotein form of human albumin. The bovine
albumin is preferably highly pure and essentially a
non-glycoprotein devoid of substantial amount of acceptor glycan
contamination.
[0110] In a preferred embodiment the non-glycosylated human serum
albumin is purified from a blood-derived human serum albumin by
affinity chromatography by a reagent binding to the glycans of
glycosylated isoform of human serum albumin. In another embodiment
the residual glycosylation is removed by a chemical or enzymatic
method. In yet another preferred embodiment the albumin is
recombinant albumin produced in prokaryotic or eukaryotic cell
producing no or not substantial amount of acceptor/substrate
glycans for the glycosyltransfeases. An example of recombinant
albumin is a yeast-produced albumin (Delta/Novozyme,
Australia/Denmark).
[0111] Preferred non-glycoproteins further include transferrin
proteins not containing acceptor glycans or not containing
substantial amount of acceptor glycans. A preferred transferrin is
a recombinant transferrin or functional fragment thereof comprising
low amount of sialyl- and/or fucosyltransferase acceptor sites,
such as a recombinant transferrin lacking glycosylsite(s), more
preferably recombinant human transferrin lacking N-glycosylation
site(s) or a recombinant transferrin produced in organism producing
no N-glycans (e.g E. coli) or producing not complex type N-glycans
(e.g. yeast S. cerevisiae or fungi such as Aspergillus). In a
preferred embodiment the transferrin is highly sialylated
transferrin, especially for use as substrate of sialyltransferase,
and/or highly .alpha.6-sialylated transferrin for use as
non-glycoprotein substrate of .alpha.3-fucosyltransferase.
Preferred sources for sialylated transferrins include isolation
from serum such as human serum and eukaryotic expression and
optionally in vitro sialylation.
[0112] The non-glycoprotein protein may be used in concentration of
0.001-5% (weight/vol), preferably 0.01-2%, more preferably 0.01-1%,
more preferably 0.05-0.5%, and in a preferred embodiment with
concentration of about 0.1%. The molecular weight of the
non-glycoprotein is preferably between 10-1000 kDa, more preferably
50-300 kDa. The very small molecular weight proteins with some
glycosylation acceptor sites are used as lover w/v concentrations
(eg. 0.001-1%, more preferably 0.05-0.5% to avoid larger glycan
concentrations and reduction of glycomodification speed)
[0113] It is realized that the protein to be used may contain some
glycosylation sites due to practical production reasons. The
invention reveals that such proteins are useful for the
glycomodification according to the invention, when containing some
acceptor sites for sialyl or fucosyltransferases such as less than
an upper limit of 50 mol % of the protein amount, more preferably
less than 35%, even more preferably less than 30%, even more
preferably less than 25%, even more preferably less than 20%, even
more preferably less than 15%, even more preferably less than 10%,
most preferably less than 5%. Because of practical reasons the
amount of acceptor sites could possibly not be limited below about
0.01%, more preferably above 0.1% even more preferably above 1%.
The practical acceptor site levels are thus range being from, e.g.,
0.01-50 mol %, 0.01-35% or 0.01-5 mol % (the lower limits are
combinable any upper limit above), of the accepor sites or in more
preferred practical range about 0.1-50 mol %, 0.1-35% or 0.1-5 mol
% or 1-50 mol %, 1-35 % or 1-5 mol %.
[0114] The preferred non-glycoproteins being not substrates for
sialylation experiment of the present invention includes proteins
which are sialylated to high level. When the protein contains high
sialic acid level, it cannot be further sialylated. A preferred
highly sialylated N-glycoprotein contains at least same amount of
monosialylated and disialylated N-glycans, more preferably at least
2 fold more disialylated biantennary N-glycans than monosialyalted
biantennary glycans, more preferably 3 fold, even more preferably 4
fold more, even more preferably 5 fold and most preferably. The
amount of non-sialylated (neutral) complex type with terminal
N-acetyllactosamine acceptor for the sialyltransferase, e.g. type
II N-acetyllactosamine, is less than 30 mol % less than 20 mol % of
the total N-glycans, more preferably less than 10%, even more
preferably less than 5%, even more preferably less than 3%, most
preferably less than 1%.
[0115] A preferred sialylated transferrin, preferably
.alpha.6-sialylated human transferrin contains practially no
neutral N-glycans, less than 5%, even more preferably less than 3%,
most preferably less than 1% and the amount of disialylated
biantennary N-glycans is at least 4 fold more, more preferably at
least 5 fold, even more preferably 6 fold more than monosialyalted
biantennary glycans.
[0116] Example 19 shows a cell culture medium protein, such as
human serum transferrin, containing high amounts diasialylated
biantennary N-glycans corresponding to monosaccharide compositions
S2H5N4 and S2H5N4F (S is Neu5Ac, H corresponds to 2 Gal and 3 Man
residues and N is GlcNAc of complex type N-glycan), being much more
abundant than corresponding monosialylated glycans S1H5N4, and
S1H5N4F1.
[0117] Limited and Controlled Amount of Glycan Acceptor
[0118] The invention reveled glycoproteins essentially devoid of
glycan acceptors or glycosylatable acceptors under the specific
reaction conditions, these includes specifically controlled and
limited acceptor glycans. Most surprisingly the present invention
reveals that it is possible to produce glycomodification conditions
where the cells are modified in the presence of amount of
glycoprotein comprising limited and controlled actual
acceptor(substrate) glycans for the glycomodification enzymes. The
limited and controlled glycan amount can comprise the substrate
glycan in excess, even large excess to the cellular glycans and
surpringly the invention revealed that cells were effectively
glycosylated.
[0119] The preferred controlled and limited acceptor glycan is
controlled with regard to glycan valency and or acceptor site
valency on the glycans, preferably there is 1-5 glycans (not all
glycosylation sites are necessarily used for every protein) maximum
5 glycans per protein, more preferably 1-4 glycans, more preferably
1-3 glycans and most preferably 1 or 2 glycans, and in specific
embodiment tonly 1 glycans, preferably the glycans are modified not
to be substrates of the specific glycmodification so the
glycosylation site valency is above 0.05 or 0.1 of the all
glycosylation sites of the glycans but on average less than 2, more
preferably about 1 or less, more preferably less than 0.3 (30%),
more preferably less than 0.2 (20%), and most preferably less than
0.1 (10%) (or other preferred valensies described by the
invention). In a preferred embodiment the amount of at least two
acceptor site containing glycans (such as neutral biantenanry
N-glycans for sialylation of fucosylation) is very low, preferably
below 20%, 10%, 5%, 3% or 1% with increasing preference.
[0120] In preferred embodiment the glycosylation modification is
preformed to cell materials on which the specific product glycan is
present in low proportion of the cells in the beginning of the
reaction (e.g. 0-50%, more preferably 1-35%, or under specific
embodiment 1-10%) but the acceptor glycan is present in substantial
part of the cells. It is realized that the high concentration of
the acceptor glycans on cell surface support the reactions.
[0121] An example of relatively large amount of limited and
controlled acceptor glycans is a medium comprising a highly
sialylated transferrin with concentration at least 0.01-0.1 mg/ml
(similar to Example 19) for modification of a million cells in
0.5-1 ml of reaction volume. There can be 100-pmol-1 nmol acceptor
glycan concentration in glycoprotein in comparision to low picomal
glycan (e.g 1-10 pmol) content of the cells. The unusual reaction
kinetics of present reactions on cell surface allows unusually
effective reactions under these conditions. The acceptor sites are
limited essentially to one site per glycan (neutral biantennary
glycan amount is low), and the valency of transferrin glycans is
maximally two, the amout of proteins with two acceptor sites per
protein on different glycans is very low (possibly 2-3%).
[0122] It is realized that it is highly suprising that the cell
surface glycosyaltion can be performed in the presence of large
proportional amount of glycan acceptor sites in comparision to the
acceptor sites on the cells, in a preferred embodiment the cells
are modified in the presence on non-glycoprotein with controlled
and limited glycosylation with at least 0.1 to about at least 1000
fold amount of acceptor sites in comparision to acceptor sites on
cells, in another embodiment the amount of sites is 1 to at least
100 fold, and in another embodiments at least 2 fold, 5 fold or 10
fold.
[0123] It is realized that due to cell specificity of the reaction
the donor nucleotide can be adjusted to low levels, this reduces
costs and amount of residual chemical in the cell preparation aimed
for biological use or it vitro studies. In another preferred
embodiment the amount of nucleotide sugar donor is adjusted level 2
times of the total acceptor site in the reaction), or 1.5 times the
total acceptor sites, or 1.1 times the total acceptor sites and
1.times. of the total acceptor site, or even 0.9, 0.8, 0.75 or 0.5
times of the acceptor sites. (total acceptor sites includes
glycoprotein and cellular acceptor sites and possible hydrolytic
activity degrading the nucleotide sugar during the reaction).
[0124] The presence of glycoproteins is of benefit for supporting
cells (e.g. transferrin), stabilizing enzymes and further with
effects preventing cell adherence.
[0125] Equivalent Ion Conditions with Single Cation of
Invention
[0126] It is realized that skilled person may optimize Mg2+ or Ca2+
concentration to allow use of only one of the cations and obtain
similar or almost similar glycomodification and in another
embodiment optionally including additional divalent cation under
non-toxic or harmful concentration. Such conditions within the
preferred or suitable to cells concentration ranges are within the
scope of the present invention
[0127] Glycomodification of Cells in the Presence of Metabolic
Supporting Factors
[0128] The invention revealed that it is possible to perform
enzymatic glycomodification in the presence of "supporting
factors", meaning here, for example, (i) vitamins, including enzyme
cofactors, (ii) nutrients with energy functions, or (iii) nutrients
with structural functions, all preferably in concentrations similar
to those in the .alpha.-MEM medium.
[0129] In a preferred embodiment the reaction is performed in the
presence of metabolic factors as present in a serum-free cell
culture medium. In a preferred embodiment the cell culture medium
is .alpha.-MEM for human mesenchymal stem cells. The medium
contains only non-glycoproteins according to the invention and no
other acceptor glycans or oligosaccharides, which would affect the
activity of the modification enzymes.
[0130] Prevention of Adherence of Novel Cells
[0131] The present cells are adherent cells and modified in a
condition preventing adhesion of the cells, especially their
clustering and surface adherence. Preferably conditions are shear
force involving conditions. It is further realized that the
prevention of clustering may be obtained by various chemical
factors.
[0132] In a preferred embodiment the shear force to the cells is
provided by tubing or tubing with a changing flow velocity that
detaches the cells, or by tubing comprising a narrowing that
provides turning flow direction and a swirl or swirls that detach
cells from each other, or by applying a flow of liquid with swirls
on cells. The administration of the shear force preferably includes
providing a certain flow accelerating and decelerating flow
velocity to the cells in the tubing (Example 2).
[0133] Increased Modification Time
[0134] The preferred method for the production of the cells
included a modification step, wherein the cells are modified for at
least 0.5 hours, more preferably over 1 hour, even more preferably
over 1.5 hours and even more preferably 2 hours or longer, such as
2 to 4 hours.
[0135] It is realized that present reaction conditions allow
maintaining the reactions over one hour, while an incubation of
stem cells in salt solutions without divalent cations leads to
apparently harmful morphological changes already within one hour
(Example 1).
[0136] Glycan Modification Including Shear Force
[0137] The invention is in a preferred embodiment directed to
modification of adherent cells including at least one step of
exposing cells to shear force. It is realized that the cells may
adhere to each other and/or cell culture support during the
modification reagent, and this would reduce the effect of the cell
modification reaction(s) and may diminish their viability (Example
2).
[0138] More preferably cells are exposed to shear forces for
maintaining unicellular cell state in presence of divalent cations,
preferably Ca.sup.2+ and/or Mg.sup.2+. It is realized that the
presence of divalent cations increase cell adhesion by the
receptors dependent on divalent cations.
[0139] In a preferred embodiment the cells are exposed to the shear
stress for at least one period during the modification reaction for
time allowing suspension of cell starting to adhere to suspend to
unicellular composition. In a preferred embodiment the shear stress
is administered for at least three, and more preferably at least
four periods. In preferred embodiment the length of the period is
at least 5 seconds, more preferably at least 15 seconds, and most
preferably at least 30 seconds.
[0140] In preferred embodiment the shear force is administered by
mechanical agitation of the cell preparation in solution. The
mechanical agitation includes administration of the cells through a
hole or tube, which is narrow enough to provide shear stress to
cells. More preferably the shear stress is administered by sucking
cells into a tip structure similar to pipette tip, preferably a tip
of a Biohit m1000 pipette with Art 1000E filter tip (Example
2).
[0141] Reaction Time and Enzyme Concentration
[0142] The invention revealed that optimal reaction can be obtained
by using proteolytic enzyme in relatively large concentrations and
amount per cells (Example 3).
[0143] Spontaneous Glycomodification after Trypsin Treatment
[0144] Spontaneous cell modification was observed when
trypsin-treated cells were incubated with the non-toxic divalent
cations and additional beneficial factors according to the
invention (Example 1).
[0145] It is realized that the prior art conditions of Sackstein
did not reveal spontaneous cell modification according to the
present invention, especially spontaneous synthesis of fucosylated
structures. Apparently, although the cells are viable under the
non-divalent cation conditions of Sackstein, they are not
biosynthetically fully active. Furthermore the status of the cells
under these conditions is so compromised that the longer reactions
appear not possible without additional damage to cells. This may
seriously limit the therapeutic use of this method.
[0146] The present conditions may be obvious for maintaining
metabolic activity of the cells, but the increase of sialyl-Lewis x
structures (Example 1), especially the core 2 sialyl-Lewis x
epitope was not expected. It is realized that the spontaneous cell
surface modification appears partially be related to glycoprotein
recovery on cells, furthermore the present media is considered to
support the machinery for endogeneous glycosylation.
[0147] The present invention is further directed to use of the
spontaneous modifications together with in vitro modification of
the cells.
[0148] Modification Conditions for Mesenchymal Stem Cells
[0149] Preferably mesenchymal stem cells are modified to increase
sialylation and/or fucosylation as a combination method, preferably
by preferred transferases for sialyl-LacNAc synthesis and
fucosylation such as STGalIII and Fuc-TVI.
[0150] Preferred fucosyltransferase conditions include about 4 mU
(especially for Fuc-TVI, Calbiochem enzyme and units, fresh enzyme)
per 3 million cells, or from 0.5 to 5 mU per million cells, more
preferably 0.75-3 mU, and most preferably 1-2 mU per million cells.
The preferred range depends on the status of the enzyme (dacays
during storage) and status and type of the cells. The preferred
reaction temperature is about 37.degree. C., preferably between
33-40.degree. C. and more preferably 35-39.degree. C. The preferred
reaction times varies from 0.5 to 6 hours preferably between 1-6
hours, more preferably between 2-6 hours, even more preferably
between 3-5.5 hours and in a preferred embodiment about 4 hours
(3.5-4.5 hours). It is realized that increasing the enzyme amount
reduces reaction time needed.
[0151] Preferred sialyltransferase conditions includes about 50 mU
(especially for .alpha.2,3-(N)-Sialyltransferase (Calbiochem),
Calbiochem enzyme and units, fresh enzyme) per 1 million cells, or
from 5 to 200 mU per million cells, more preferably 10-150 mU, and
most preferably 25-75 mU per million cells. The preferred range
depends on the status of the enzyme (dacays during storage) and
status and type of the cells. The preferred reaction times varies
from 0.5 to 6 hours preferably between 1-6 hours, more preferably
between 2-6 hours, even more preferably between 3-5.5 hours and in
a preferred embodiment about 4 hours (3.5-4.5 hours). The preferred
reaction temperature is about 37.degree. C., preferably between
33-40.degree. C. and more preferably 35-39.degree. C. It is
realized that increasing the enzyme amount reduces reaction time
needed.
[0152] The invention is especially directed to modification of stem
cells especially mesenchymal stem cells wherein the cells have
unusually low sialylation levels. The cells with low sialylation
comprise more than 30% of N-glycans in non-sialylated form. In a
preferred embodiment the mesenchymal stem cell with low sialylation
is a bone marrow derived mesenchymal stem cell (Example 6, Example
9).
[0153] Preferred Glycosylation Levels for Modification of Cells
[0154] The present invention revealed that it is possible to
glycosylate cells, preferably to sialylate cells to over 50% level
of available free sialylation sites on N-glycans (when calculated
based on the disappearance of the sialylation sites). In a
preferred embodiment the invention is directed to sialylation by
single sialyltransferase to level over 60%, more preferably over
70%, even more preferably over 75%, even more preferably over 80%,
or at least 83%, and most preferably over 85%. The invention is
further directed to a novel mesenchymal stem cell population
comprising increased sialylation of over 60%, more preferably over
70%, even more preferably over 75%, even more preferably over 80%,
or at least 83%, and most preferably over 85%. The cell population
is preferably derived from human cord blood or bone marrow.
[0155] Characteristics of Novel Cells
[0156] Viable, Intact Cells
[0157] The invention is directed to the novel cells, wherein the
cells are at least 98% viable as indicated by intact plasma
membrane, most preferably over 99% as indicated by intact plasma
membrane. The invention revealed that under the preferred cell
modification condition, including e.g. non-toxic cations, leads to
highly viable cells, especially, when the cells are produced under
conditions in which adherence is actively inhibited by shear force.
The preferred viable mesenchymal cells according to the invention
have intact plasma membrane (Example 2).
[0158] The invention further revealed that the control cells did
suffer from the incubation in the buffer without the beneficial
reagent according to the present invention. It is realized that
though the control cells have mostly intact membranes their overall
viability and normal cell status is compromised.
[0159] Preferred Markers of Protease Produced Glycan Modified
Cells
[0160] The invention revealed referred markers of novel glycan
modified cell populations, preferably protease produced glycan
modified cell populations. The invention is especially directed to
in vitro fucosylation methods, when fucosylation increases amount
of core 2 sLex, especially epitope similar to one recognized by
antibody CHO-131. This structure is effectively formed under
present cell modification conditions. The optimal production is
obtained by spontaneous regeneration of glycosylation, preferably
fucosylation by endogenous fucosyltransferase, and/or by in vitro
glycosylation preferably fucosylation or fucosylation and
sialylation, preferably by both.
[0161] Effects of Sialylation and Fucosylation
[0162] The invention revealed that the sialylation and fucosylation
reactions according to the invention increased specific high MAA
subpopulation of the mesenchymal cells. MAA recognizes a specific
highly .alpha.3-sialylated in the novel cell populations (Example
1). The fucosylation and sialylation reactions further provide
effectively the production of sialyl-CD15 and core 2 sLex (antibody
CHO-131) epitopes.
[0163] Cells with Lower Amount of Sialylated Glycomarkers
[0164] The invention is in a preferred embodiment directed to
modification of specific mesenchymal stem cell preparations, in a
preferred embodiment produced by protease, such as trypsin
treatment wherein the amount of certain glycan structures are
reduced (Example 1):
[0165] the amount of N-glycans, which can be modified by
sialylation on cell surface, or the amount of specific non-N-glycan
sLex (sialyl-Lewis x) epitope, preferably O-glycan associated
glycans, especially GF526 epitope (antibody clone CHO-131). The
GF526 epitope is core 2 O-glycan, comprising sLex on .beta.-linked
arm. The structure is in other context associated with PSGL-1
protein.
[0166] Quantitative Change of Sialylation Levels
[0167] The invention revealed that it is possible to change
quantitatively the sialylation levels of human cells. The signals
of monosialylated and disialylated sialic acids of biantennary
N-glycan cores were measured by MALDI-TOF mass spectrometry of
released non-modified N-glycans. It was observed that the
sialylation levels of the N-glycans of cells could be increased at
least by 15% units and even by about 20% or 25% by sialylation of
the cells by sialyltransferase enzyme. It was also observed that
the sialylation levels of the N-glycans of cells could be decreased
at least by 15% units and even by about 20% or 25% by sialylation
of the cells by sialylidase (neuraminidase) enzyme.
[0168] The invention is especially directed to cell populations of
quantitatively increased and decreased sialylation levels.
[0169] The invention revealed furthermore that the
.alpha.3-sialylated cells can be fucosylated to produce cells
increased in their sialylated and fucosylated levels, comprising
sialyl-Lewis x Neu5Ac.alpha.3Ga.beta.4(Fuc.alpha.3)GlcNAc (sLex)
and related structures. It is realized that sLex content can be
further increased by first resialylating the cells and thus
reducing .alpha.6-sialylated structures blocking sites. Such
sialyl-Lewis x cells are especially useful for in vivo targeting as
the structures produced in low amounts from endogenous
Neu5Ac.alpha.3Gal.beta.4GlcNAc can redirect the cells (Xia et al.
2004) (Example 1).
[0170] Biological Use of the Modified Cells
[0171] It is realized that the novel cells produced by the
invention are useful for in vivo targeting in human and animal
trials, or potentially in therapeutic applications.
[0172] The invention is in a specific embodiment directed to
altering surface adhesion of glycan modified cells. It is thus
realized that glycan modification can be used to change adherence
properties of cells. The change of adherence properties is used for
e.g. [0173] For in vivo targeting methods such as targeting cells
to a particular tissue, as described by Sackstein and colleagues;
[0174] In vitro methods requiring altered cell adherence such as
attaching cells for detection of cells, in a preferred embodiment
as adherent cells e.g. for microscopy or as soluble cells e.g. in
flow cytometry or use of the adherent or soluble cells for cell
adhesion studies, preferably for adhesion studies with lectins such
as selectins; [0175] Ex vivo cell culture methods producing
transient cell modifications for subsequent in vivo targeting.
[0176] The invention is in a specific embodiment directed to cell
culture of glycan modified cells with altered adherence properties.
In a preferred embodiment the cells are cultured after
modification, meaning increase of sialylation by sialyltransferase
or reduction of sialic acids by sialidase enzyme. In a preferred
embodiment the invention is directed to modification of naturally
adherent cells, preferably mesenchymal stem cells.
[0177] Refinements of the Present Invention
[0178] Removable Enzymes by Tagging
[0179] The invention revealed novel effective methods for modifying
cells by glycosyl modifying enzymes such as glycosidases and/or
glycosyltransferases, when the enzymes are removed from the cell
preparations. The invention is especially directed to use of
specific tag-structures for the removal of the enzymes from the
cells (Example 5).
[0180] Release of Enzymes by Carbohydrate Enzyme Inhibitors
[0181] It is realized that enzymes bind cells by their carbohydrate
binding sites such as catalytic sites. In another embodiment the
enzymes are removed by incubating the cells with an inhibitor of
the enzymes, preferably an inhibitor binding to the catalytic
carbohydrate recognizing site of the enzyme. Preferred inhibitors
include monosaccharides and monosaccharide glycosides such as
methyl and ethyl glycosides and more specific inhibitors, which may
be designed based on the catalytic site as transition state
inhibitors. Preferred inhibitors for sialidases include competitive
low activity inhibitors such as sialic acid, and modified or low
cost competing substrates such as NeuAc.alpha.OMe,
NeuNAc.alpha.OEt, sialyl-Lactoses available e.g. from bovine milk
or polysialic acid available from bacteria (E. coli, colomnic
acid): and higher activity inhibitors such as NeuAc2en (NeuNAc with
double bond between 2- and 3-positions) or e.g. higher activity
inhibitors specific for limited number of enzymes such as influenza
virus neuraminidase inhibitors: Tamiflu (oseltamivir, Roche) or
Zanamivir (GSK).
[0182] The amount of enzyme inhibitor needed can be estimated by
inhibition constants. Competitive monosaccharide glycoside or
oligosaccharide inhibitors with low millimolar inhibition (or
binding constants) are typically needed in amounts of several fold
or order of magnitude larger amounts than the inhibition constant.
Typical concentrations for the low affinity inhibitors are of about
1-500 mM, more preferably 1-250 mM, and more preferably 2-100 mM,
or 2 to 50 mM, even more preferably from about 2 mM to 20 mM. The
lower ranges are preferred to maintain the stability and osmotic
condition of the cells stable. Typical concentrations for higher
affinity inhibitors are from about 1 pM to about 10 mM, depending
about the affinity constants. Preferred concentrations for low
range micromolar inhibitor are between 10-1000 micromolar. Suitable
inhibition concentrations are available from literature.
[0183] The invention is directed for removing modification enzyme
from modified cells involving a step of incubation of the cells
with an inhibitor or substrate of the enzyme. The method preferably
further comprises steps of washing cells with a suitable solution
such as PBS (phosphate buffered saline) or other solution suitable,
optionally containing additional amount of inhibitor, and
preferably a step of final washing with the solution not comprising
the inhibitor.
[0184] In an embodiment the invention is directed to removal of the
enzyme by a combination of the enzyme tagging with the use of the
inhibitors.
[0185] Specifications of Glycomodifications
[0186] Desialylation Methods
[0187] The invention is specifically directed to desialylation
methods for modification of human cord blood cells. The cord blood
cells are clearly different of other cell types and no
desialylation methods have previously been developed for these
cells. Due to cell specific differences any quantitative
desialylation methods cannot be generalized from one cell
population to another. Thus, any results and data demonstrated by
other investigators using other cell types are not applicable to
cord blood. The present invention is further directed to
desialylation modifications of any human stem cell or cord blood
cell subpopulation.
[0188] The invention is preferably directed to linkage specific
.alpha.3-desialylation of the preferred structures according to the
invention without interfering with the other sialylated structures
according to the present invention. The invention is further
directed to simultaneous desialylation .alpha.3- and
.alpha.6-sialylated structures according to the present
invention.
[0189] Furthermore the present invention is directed to
desialylation when both NeuAc and
[0190] NeuGc are quantitatively removed from cell surface,
preferably from the preferred structures according to the present
invention. The present invention is specifically directed to the
removal of NeuGc from preferred cell populations, most preferably
cord blood and stem cell populations and from the preferred
structures according to the present invention.
[0191] Modification of Cell Surfaces of the Preferred Cells by
Glycosyltransferases
[0192] The inventors revealed that it is possible to produce
controlled cell surface glycosylation modifications on the
preferred cells according to the invention.
[0193] The present invention is directed to cell modifications by
sialyltransferases and fucosyltransferases. Two most preferred
transfer reactions according to the invention are
.alpha.3-modification reactions such as .alpha.3-sialylation and
.alpha.3-fucosylations. When combined these reactions can be used
to produce important cell adhesion structures which are sialylated
and fucosylated N-acetyllactosamines such as sialyl-Lewis x
(sLex).
[0194] Possible .alpha.6-sialylation has been implied in bone
marrow cells and in peripheral blood CD34+ cells released from bone
marrow to circulation by growth factor administration, cord blood
cells or other stem cell types have not been investigated.
Furthermore, the previous study utilized an artificial sialic acid
modification method, which may affect the specificity of the
sialyltransferase enzyme and, in addition, the actual result of the
enzyme reaction is not known as the reaction products were not
analysed by the investigators. The reactions are likely to have
been very much limited by the specificity of the
.alpha.6-sialyl-transferase used and cannot be considered prior art
in respect to the present invention.
[0195] The inventors of the present invention further revealed
effective modification of the preferred cells according to the
present inventions by sialylation, in a preferred embodiment by
.alpha.3-sialylation.
[0196] The prior art data cited above does not indicate the
specific modifications according to the present invention to cells
from early human blood, preferably cord blood, to cultured
mesenchymal stem cells, or to cultured embryonal type cells. The
present invention is specifically directed to sialyltransferase
reactions towards these cell types. The invention is directed to
sialyltransferase catalyzed transfer of a natural sialic acid,
preferably NeuAc, NeuGc or Neu-O--Ac, from CMP-sialic acid to
target cells.
[0197] Sialyltransferase catalyzed reaction according to
Formula:
CMP-SA+target cell SA.fwdarw.target cell+CMP,
[0198] Wherein SA is a sialic acid, preferably a natural sialic
acid, preferably NeuAc, NeuGc or Neu-O-Ac and
[0199] the reaction is catalysed by a sialyltransferase enzyme
preferably by an .alpha.3-sialyltransferase and
[0200] the target cell is a cultured stem cell or early human blood
cell (cord blood cell).
[0201] Preferably the sialic acid is transferred to at least one
N-glycan structure on the cell surface, preferably to form a
preferred sialylated structure according to the invention.
[0202] In the prior art fucosyltransferase reactions towards
unspecified cell surface structures have been studied. The prior
art indicates that human cord blood cell populations may be
.alpha.3-fucosylated by human fucosyltransferase VI and such
modified cell populations may be directed to bone marrow due to
interactions with selectins.
[0203] Methods for combined increased .alpha.3-sialylation and
.alpha.3-fucosylation
[0204] The invention is specifically directed to selection of a
cell population from the preferred cell population according to the
present invention, when the cell population demonstrate increased
amount of .alpha.3-sialylation when compared with the baseline cell
populations.
[0205] The inventors revealed that human cord blood in general is
highly .alpha.6-sialylated and thus not a good target for
.alpha.3/4-fucosylation reactions, especially for reactions
directed to production of selectin ligand structures.
[0206] Fucosylation of .alpha.3-sialylated cells
[0207] The present invention is preferably directed to fucosylation
after .alpha.3-sialylation of cells, preferably the preferred cells
according to the invention. The invention describes for the first
time combined reaction by two glycosyltransferases for the
production of specific terminal epitopes comprising two different
monosaccharide types on cell surfaces.
[0208] Production of Preferred Sialylated Structures
[0209] Present invention is specifically directed to methods for
sialylation to produce preferred structures according to the
present invention from the surfaces of preferred cells. The present
invention is specifically directed to production preferred NeuGc-
and NeuAc-structures. The invention is directed to production of
potentially in vivo harmful structures on cells surfaces, e.g. for
control materials with regard to cell labelling. The invention is
further directed to production of specific preferred terminal
structure types, preferably .alpha.3- and .alpha.6-sialylated
structures, and specifically NeuAc- and NeuGc-structures for
studies of biological activities of the cells.
[0210] The present invention is further directed to preferred
methods for the quantitative verification of the sialylation by the
preferred analysis methods according to the present invention. The
present invention is further directed to linkage specific
sialylation and analysis of the linkage specific sialylation on the
preferred carbohydrate structures using analytical methods
according to the present invention.
[0211] The invention is preferably directed to linkage specific
.alpha.3-sialylation of the preferred structures according to the
invention without interfering with the other sialylated structures
according to the present invention. The invention is preferably
directed to linkage specific .alpha.6-sialylation of the preferred
structures according to the invention without interfering with the
other sialylated structures according to the present invention.
[0212] The invention is further directed to simultaneous
sialylation .alpha.3- and .alpha.6-sialylated structures according
to the present invention. The present invention is further directed
for the production of preferred relation of .alpha.3- and
.alpha.6-sialylated structures, preferably in single reaction with
two sialyl-transferases.
[0213] Furthermore the present invention is directed to sialylation
when either NeuAc or NeuGc are quantitatively synthesized to the
cell surface, preferably on the preferred structures according to
the present invention. Furthermore the invention is directed to
sialylation when both NeuAc and NeuGc are, preferably
quantitatively, transferred to acceptor sites on the cell
surface.
[0214] The present invention is specifically directed to the
removal of NeuGc from preferred cell populations, most preferably
cord blood cell populations and from the preferred structures
according to the present invention, and resialylation with
NeuAc.
[0215] The invention is further directed to preferred methods
according to the present invention for verification of removal of
NeuGc, and resialylation with NeuAc, preferably quantitative
verification and more preferably verification performed by mass
spectrometry with regard to the preferred structures.
[0216] Controlled Cell Modification
[0217] The present invention is further directed to cell
modification according to the invention, preferably desialylation
or sialylation of the cells according to the invention, when the
sialidase reagent is a controlled reagent with regard of presence
of carbohydrate material.
[0218] Purification of Cells with Regard to Modification Enzyme
[0219] The preferred processes according to the invention comprise
of the step of removal of the enzymes from the cell preparations,
preferably the sialyl modification enzymes according to the
invention. Most preferably the enzymes are removed from a cell
population aimed for therapeutic use. The enzyme proteins are
usually antigenic, especially when these are from non-mammalian
origin. If the material is not of human origin its glycosylation
likely increases the antigenicity of the material. This is
particularily the case when the glycosylation has major differences
with human glycosylation, preferred examples of largely different
glycosylations include: prokaryotic glycosylation, plant type
glycosylation, yeast or fungal glycosylation, mammalian/animal
glycosylation with Gal.alpha.3Gal.beta.4GlcNAc-structures, animal
glycosylations with NeuGc structures. The glycosylation of a
recombinant enzyme depends on the glycosylation in the production
cell line, these produce partially non-physiological glycan
structures. The enzymes are preferably removed from any cell
populations aimed for culture or storage or therapeutic use. The
presence of enzymes which have affinity with regard to cell surface
may otherwise alter the cells as detectable by carbohydrate binding
reagents or mass spectrometric or other analysis according to the
invention and cause adverse immunological responses.
[0220] Under separate embodiment the cell population is cultured or
stored in the presence of the modification enzyme to maintain the
change in the cell surface structure, when the cell surface
structures are recovering from storage especially at temperatures
closer physiological or culture temperatures of the cells.
Preferably the cells are then purified from trace amounts of the
modification enzyme before use.
[0221] The invention is furthermore directed to methods of removal
of the modification reagents from cell preparations, preferably the
modification reagents are desialylation or resialylation reagents.
It is realized that soluble enzymes can be washed from the modified
cell populations. Preferably the cell material to be washed is
immobilized on a matrix or centrifuged to remove the enzyme, more
preferably immobilized on a magnetic bead matrix.
[0222] However, extraneous washing causes at least partial
destruction of cells and their decreased viability. Furthermore,
the enzymes have affinity with regard to the cell surface.
[0223] Therefore the invention is specifically directed to methods
for affinity removal of the enzymes. The preferred method includes
a step of contacting the modified cells with an affinity matrix
binding the enzyme after modification of the cells.
[0224] Under specific embodiment the invention is directed to
methods of tagging the enzyme to be removed from the cell
population. The tagging step is performed before contacting the
enzyme with the cells. The tagging group is designed to bind
preferably covalently to the enzyme surface, without reduction or
without major reduction of the enzyme activity. The invention is
further directed to the removal of the tagged enzyme by binding the
tag to a matrix, which can be separated from the cells. Preferably
the matrix comprises at least one matrix material selected from the
group: polymers, beads, magnetic beads, or solid phase surface.
[0225] Tagging of Enzyme for Modification Glycan Controlled
Enzymes
[0226] The invention is furthermore directed to methods of removal
of the modification reagents from cells to be depleted of sialic
acid and/or resialylated. The preferred modification reagents are
desialylation or resialylation reagents. The reagents are tagged to
be able to bind the reagents to solid phases comprising specific
binder recognizing the tag, the tag binder combination e.g. on
microbeads can be removed.
[0227] Preferred tags include
[0228] antigens such as peptide FLAG or HA-hemagglutinin peptide
tag, or
[0229] chemical tags such as His-tag or fluoroalkane or
[0230] biotin.
[0231] The invention is directed to known specific binders for
these, such as specific antibodies for peptides, his-tag binding
column for His-TAG, fluoroalkane for hydrogen bond binding of
fluoroalkane and avidin or streptavidin for biotin are used.
[0232] Preferred modification enzymes and enzymes to be tagged
include sialidase (neuraminidases) such as .alpha.3-, .alpha.6- and
multispecific sialidases and .alpha.3-, .alpha.6-sialyltransferases
for example from mammalian or bacterial origin and specific for
type I and/or type II N-acetyllactosamines, preferably type two
N-acetyllactosamines and N-glycans especially biantennary and
triantennary N-glycans known in the art. The invention is
specifically directed to preferred tagged enzymes as
substances.
[0233] Preferred bacterial fucosyltransferases include enzymes
homologous to human .alpha.3/4-fucosyltransferases, such as
Helicobacter pylori fucosyltransferases homologous to enzyme
described in Sun H-Y. J. Biol. Chem. (2007) manuscript M601285200,
published Jan. 24, 2007. The preferred specificities of bacterial
such as H. pylori fucosyltransferase include reaction with
3'modified lactosamines such as Neu5Ac.alpha.3Gal.beta.4GlcNAc to
synthesize sialyl-Lewis x, known to be produced at lest by part of
H. pylori strains.
[0234] Under specific embodiment the invention is directed to
methods of tagging the enzyme to be removed from the cells.
[0235] Preferably a sialidase enzyme or sialyltransferase is linked
to tag-molecule, the tagged enzyme is reacted with the cells to be
remodelled and the enzyme is removed after the reaction by
immobilizing the enzyme by binding to a molecule specifically
binding to the tag and the modified cell(s) are removed from the
immobilized enzyme by filtering the cells with matrix of a molecule
specifically binding to the tag, preferred matrices include column
used for cell purification or magnetic beads used for purification
of components from cell mixtures (see protocols or catalogs of
Dynal and Miltenyi companies).
[0236] The tagging step is preferably performed before contacting
the enzyme with the cells. The tagging group is designed to bind
preferably covalently to the enzyme surface, without reduction or
without major reduction of the enzyme activity. Preferred covalent
linkage occurs to amine groups, thiol group or oxidized glycan
groups as known from catalogue of Pierce.
[0237] The invention is further directed to the removal of the
tagged enzyme by binding the tag to a matrix, which can be
separated from the cells to be modified. Cells proteins are
preferably separated from tag-binder immobilized reagents in
aqueous media as known in the art of using the tags. Preferably the
matrix comprises at least one matrix material selected from the
group: polymers, beads, magnetic beads, or solid phase surface.
[0238] Enzymes Acceptable for Humans for Modification of Reagents
or Cells
[0239] Under specific embodiment the invention is directed to the
use for modification of the cells according to the invention, or in
a separate embodiment reagents for processes according to the
invention, of a human acceptable enzyme, preferably sialidase or
sialyltransferase, which is acceptable at least in certain amounts
to human beings without causing harmful allergic or immune
reactions. It is realized that the human acceptable enzymes may not
be needed to be removed from reaction mixtures or less washing
steps are needed for desirable level of the removal. The human
acceptable enzyme is in preferred embodiment a human
glycosyltransferase or glycosidase. The present invention is
separately directed to human acceptable enzyme which is a
sialyltransferase. The present invention is separately directed to
human acceptable enzyme which is a sialidase, the invention is more
preferably directed to human sialidase which can remove specific
type of sialic acid from cells.
[0240] In a preferred embodiment the human acceptable enzyme is
purified from human material, preferably from human serum, urine or
milk. In another preferred embodiment the enzyme is recombinant
enzyme corresponding to natural human enzyme. More preferably the
enzyme corresponds to human natural enzyme corresponds to natural
cell surface or a secreted from of the enzyme, more preferably
serum or urine or human milk form of the enzyme. Even more
preferably the present invention is directed to human acceptable
enzyme which corresponds to a secreted form of a human
sialyltransferase or sialidase, more preferably secreted
serum/blood form of the human enzyme. In a preferred embodiment the
human acceptable enzyme, more preferably recombinant human
acceptable enzyme, is a controlled reagent with regard to potential
harmful glycan structures, preferably NeuGc-structures according to
the invention. The recombinant proteins may contain harmful
glycosylation structures and inventors revealed that these kinds of
structures are also present on recombinant glycosyltransferases,
even on secreted (truncated) recombinant glycosyltransferases.
[0241] Quantitative and qualitative mass spectrometric analysis of
modified cells and or reagents The present invention is further
directed to the quantitative and qualitative mass spectrometric
analysis of modified cells and/or reagents according to the
invention.
[0242] The invention is directed to production of qualitative
glycome analysis of the cell and/or the reagents including
determining the monosaccharide composition obtained for the
materials.
[0243] The present invention is further directed to quantitative
mass spectrometric analysis of the materials according to the
invention involving determining the intensities of all or part of
the mass spectrometric signals verified to be (reasonably)
quantitative with regard to the amount of molecules corresponding
to the signals, preferably MALDI-TOF mass spectrometric
signals.
[0244] The invention is further directed to methods, especially
research an development methods, such as product development
methods, according to the invention for production of reagents or
cells as described by the invention involving step of quantitative
and/or qualitative glycome analysis, more preferably both
quantitative and qualitative analysis.
[0245] Preferred Glycan Controlled Reagents and Processes for
Preparation Thereof
[0246] Preferred reagents to be controlled include preferably all
reagents derived from or produced in connection with biological
material; preferably these include all glycoprotein, protein
mixture, serum, and albumin preparations present in the process.
The inventors found out that albumins known to be non-glycosylated
proteins may still contain sufficient glycoproteins for
contamination of cell material.
[0247] In a preferred embodiment the present invention is directed
to the control of animal albumins, preferably bovine serum albumin,
and human serum albumin preparations for potential contamination by
glycan structures.
[0248] Other preferred controlled reagents include controlled
transferrin and other serum proteins, even more preferably
controlled serum proteins are controlled antibody preparations,
preferably Fc blocking antibody preparations.
[0249] In yet another embodiment the invention is directed to the
production of glycan depleted and/or remodelled protein mixtures
preferably glycan remodelled human or animal serum, more preferably
a serum from an animal used for production of serum products,
preferably cell culture serum or antibodies. Preferred serums to be
modified includes serum of cow, horse, sheep, goat, rabbit, rat or
mouse, more preferably serum of cow, horse, or sheep, even more
preferably fetal bovine serum.
[0250] In a preferred embodiment the glycosylation of the serum is
altered by a method based on animals with genetically altered
glycan production preferably obtained by a) genetic manipulation of
the animal or b) breeding a natural or selecting a natural variant
of the production animal to used for serum production, preferably
the genetic alteration is directed to tissues producing serum
proteins.
[0251] Controlled Enzyme Preparations for Products Aimed for Use
with Transplantable Cells
[0252] The present invention is directed under specific embodiment
to methods for removal of non-desired carbohydrate structures from
living cells. The enzyme proteins are usually antigenic, especially
when these are from non-mammalian origin, such as bacteria and/or
plants. If the material is not of human origin its glycosylation
likely increases the antigenicity of the material. This is
particularly the case when the glycosylation has large differences
with human glycosylation, preferred examples of largely different
glycosylations include: prokaryotic glycosylation, plant type
glycosylation, yeast or fungal glycosylation, mammalian/animal
glycosylation with Gal.alpha.3Gal.beta.4GlcNAc-structures, animal
glycosylation with NeuGc structures. The glycosylation of a
recombinant enzyme depends on the glycosylation of the production
cell line, these produce partially non-physiological glycan
structures in most cases.
[0253] Preferred Classes of Controlled Reagents
[0254] Glycan Depleted Biological Materials, Preferably
Glycoprotein Materials
[0255] Present invention is specifically directed to use biological
materials, preferably glycoprotein material, from which harmful
structure is removed or reduced in amount. Glycoproteins are major
source of bioactive glycans, in some material presence of
glycolipids may be also possible and could be handled similarly. In
case the lipid part of glycolipid binds it to the material,
released glycan or part of it is water soluble and can be
separated. The invention is further directed to glycan depletion
methods. In a preferred embodiment the invention is directed to
methods including steps of releasing glycan structure and removing
released glycan structure.
[0256] Preferred methods for removal of the released glycan
structure include filtration methods. The filtration methods are
based on size difference of the released glycan structure and the
glycan depleted protein. A preferred method for removal of the
released glycans includes precipitation methods, in a preferred
embodiment the invention is directed to precipitation of the
protein under conditions where the released glycan structure is
soluble.
[0257] The glycan depletion may be combined with a step of
inactivation of potential harmful proteins such as lectins or
antibodies possibly involved in the process. Some reagents such
serum in certain cell culture processes may be heat inactivated.
The inactivation may be partial. The partial inactivation is in a
preferred embodiment performed by releasing glycans inhibiting the
harmful binding proteins to the reagent and further to cell
involving process. In a preferred embodiment the depleted glycan
and the binding protein inhibiting glycan is the same structure.
Preferably the released glycans are used when these can not be
incorporated to cells to cause further problems in the cell related
process. The method of released glycans is not preferred for NeuGc
under conditions where it can be incorporated to cells.
[0258] Terminally Depleted Glycans
[0259] In a preferred embodiment one or several terminal structures
are depleted from a biological material, preferably glycoprotein
material. The preferred methods to deplete terminal structures
include enzymatic and chemical methods. Preferred enzymatic method
is hydrolysis by a glycosidase enzyme or by a trans-glycosylating
enzyme capable of removing the terminal structure. Terminal
depletion may further include release of several terminal
monosaccharide units for example by glycosidase enzymes. Preferred
chemical hydrolysis is an acid hydrolysis, preferably a mild acid
hydrolysis under conditions not destroying protein structure or
from which the protein structure can be restored or renatured. The
structure to be depleted is in a preferred embodiment a sialic
acid. The sialic acid is preferably released by a sialidase enzyme
or by mild acid hydrolysis.
[0260] Internally Depleted Glycans
[0261] The present invention is further directed to internal
depletion of glycan material by release of glycans from subterminal
linkages by chemical and/or enzymatic methods. Methods to release
glycans chemically include base hydrolysis methods such as beta
elimination for release of O-linked glycans, hydrazinolysis methods
to release O-glycans and N-glycans, oxidative methods such as Smith
degradation and ozonolysis (preferred for glycolipids). Preferred
enzymatic methods include use of endo-glycosidases such as
endoglycosylceramidase for glycolipids, N-glycosidases for
N-glycans, and O-glycosidases for O-glycans.
[0262] Glycosylated Reagents from Non-Animal Sources
[0263] In a preferred embodiment the present invention is directed
to the use of reagents from non-animal sources devoid of
potentially harmful reagents. Preferred non-animal glycosylated
proteins are proteins from yeasts and fungi and from plants. It is
notable that even these materials contain glycans, which may have
harmful allergenic activities or which may cause problems in
analysis of human type glycans. Preferably the invention is further
directed to control of the glycosylated reagents from non-animal
structures, too. Preferred plant derived proteins include
recombinant albumins produced by plant cell culture, more
preferably non-glycosylated human serum albumins and bovine serum
albumins and recombinant gelatin materials such as collagens
produced by plant cell systems. The present invention is
specifically directed to the processes according to present
invention, when a material containing glycans or harmful glycans
according to the present invention is replaced by a reagent,
preferably a controlled reagent from non-animal sources.
[0264] Non-Glycosylated Reagents from Prokaryotes
[0265] Many bacterial recombinant proteins are known for lacking
expression of glycans. Present invention is directed to control of
glycosylation of bacterial protein, as this happens on certain
proteins. The present invention is specifically directed to the
processes, when a material containing glycans or harmful glycans
according to the present invention is replaced by a reagent,
preferably a controlled reagent from prokaryotes.
[0266] Under specific embodiment the present invention is directed
to use of glycan controlled forms of glycosidase enzymes for
modification of transplantable cells according to the invention and
removal of the enzymes from reactions as described by the present
invention
[0267] The present invention is also specifically directed to the
glycan controlled enzyme preparations, especially when produced in
a mammalian cell line/cultivation process and controlled with
regard to Gal.alpha.3Gal.beta.4GlcNAc-structures, animal
glycosylations with NeuGc structures. The preferred enzymes are of
human origin, more preferably recombinant enzymes. Most preferably
a human serum form of the enzyme is selected and the glycosylation
is controlled to be a non-antigenic human-type glycosylation,
preferably similar to the glycosylation human natural soluble
enzyme.
[0268] Preferred Sialyltransferases
[0269] Preferred sialyltransferases includes mammalian, more
preferably human .alpha.3-, and .alpha.6-sialytransferases,
preferably in soluble form. In a preferred embodiment the
transferase sialylates N-acetyllactose amines such as ST3GalIII and
ST3GalIV or ST6GalI or O-glycans core I such as ST3GalI or ST3GalII
and ST3GalIV. It is realized that most effective sialylation is
obtained with combination of at least two sialyltransferases such
as core I sialylating and N-acetyllactosamine sialylating, e.g.
ST3GalIII and ST3GalIV or ST3GalI/II and ST3GalIV.
[0270] Preferred Fucosyltransferases
[0271] Preferred fucosyltransferases include mammalian, more
preferably human .alpha.3-, and .alpha.6-fucosyltransferases,
preferably in soluble form. In a preferred embodiment the
transferase reacts with N-acetyllactosamines such as FTIII, FTIV,
FTV, FTVI, FTVII and FTIX more preferably
sialyl-.alpha.3-N-acetyllactosamines, preferably FTIII, FTIV, FTV,
FTVI, FTVII, more preferably FTIII, FTV, FTVI, and FTVII, even more
preferably FTVI, and FTVII, and most preferably FTVI. It is
realized that most effective fucosylation is obtained with
combination of at least two fucosyltransferases.
[0272] In a preferred embodiment the galactosylation reaction is
performed in the presence Mg.sup.2+ ions as described in
US2005014718, preferably by mammalian GalT, more preferably natural
human GalT, or using exogenous transferase such as Mg.sup.2+
selective .beta.4-Galactosyltransferase of Qasba and
Ramakrisnan.
[0273] It is realized that it is useful to remove exogenous GalT
and/or sialyltransferase by using specific Tags according to the
invention and/or by using enzyme inhibitors according to the
invention.
[0274] Controlled Reagents for the Modification
[0275] In another preferred embodiment it is useful to use glycan
controlled sialyltransferase or galactosyltransferase. The
invention is directed to analysis of glycans of non-human expressed
glycosyltransferases. When the transferases comprise non-human
glycosylation, the non-human structures are preferably removed by
specific glycosidases or modified by chemically e.g. by perjodate
oxidation and reduction.
[0276] The invention is especially directed to use of controlled
enzyme substances, preferably galactosyltransferase or
sialyltransferase for reaction according to the invention
comprising glycan according to Formula
[0277]
Man.alpha.6(Man.alpha.3)Man.beta.4GlcNAc.beta.4(Fuc.alpha.6)nGlcNAc-
-N-E
[0278] wherein E is enzyme protein, N is glycosidic linkage
nitrogen in N-glycosylation site (Asn-X-Ser/Thr), and the
non-reducing end mannoses may be further modified by
(NeuNAc.alpha.3/6)mGal4GlcNAc.beta.2, wherein m and n are 0 or
1.
[0279] If the enzyme would comprise NeuNGc instead of NeuNAc, this
is preferably removed and changed to NeuNAc.
[0280] In another preferred embodiment the enzymes are
non-glycosylated preferably from bacterial production e.g. as
described by Qasba US2007258986 (included fully as reference) or
N-(Asn-X-Ser/Thr/Cys) and possible O-glycosylation sites of the
enzymes are mutated for expression in eukaryotic system.
[0281] The structure is especially beneficial because
Man.beta.4-residue is devoid of Xyl.beta.2-modification present in
plant cells and reducing end GlcNAc is devoid of
Fuc.alpha.3-structure present in insect or plant cell derived
material (e.g. when the enzyme would be produced by insect or plant
cell culture).
[0282] Glycosyltransferase Inhibitors for Release of
Glycosyltransferase from Cells
[0283] The present invention is especially directed to use of
analogs or derivatives of acceptor saccharides or donor nucleotides
for inhibitors of glycosyltransferases for washing the transferase
effectively from cells after the reaction. The preferred acceptor
analogs include carbohydrates oligosaccharides, monosaccharides and
conjugates and analogs thereof capable of binding to substrate site
and inhibiting the acceptor binding of the enzyme. The preferred
concentrations of the carbohydrates includes concentrations
tolerable by the cells from 1 mM to 500 mM, more preferably 5 mM to
250 mM and even more preferably 10-100 mM, higher concentrations
are preferred for monosaccharides and method involving solid phase
bound binders.
[0284] Preferred oligosaccharide for sialyltransferase inhibition
includes sequences including oligosaccharides and reducing end
conjugates includes Gal.beta.4Glc, Gal.beta.4GlcNAc,
Gal.beta.3GlcNAc, Gal.beta.3GalNAc depending. GalT inhibitors
include GlcNAc and conjugates and GlcNAc.beta.2Man,
GlcNAc.beta.6Gal and GlcNAc.beta.3Gal.
[0285] In a preferred embodiment sialyltransferase is released by
acceptor disaccharide, more preferably by 5-150 mM acceptor, more
preferably by 10-100 mM, even more preferably 10-80 mM, more
preferably 10-50 mM for high affinity acceptor and 20-100 mM, more
preferably 40-100 mM, most preferably 50-100 mM for low affinity
acceptor. It is realized that acceptor affinities varies between
enzymes, lactose is considered as medium low affinity acceptor for
.alpha.2,3-(N)-Sialyltransferase (Calbiochem) or ST3GalIII and high
affinity acceptors have typically acceptor Km values about 10 fold
lower. Preferably washing removes at least 50% of the cell bound
enzyme even more preferably at least, 70%, even more preferably at
least 85%, even more preferably at least 90% and most preferably at
least 95%.
[0286] The preferred reducing end structure in conjugates is
[0287] AR, wherein A is anomeric structure preferably beta for
Gal.beta.4Glc, Gal.beta.4GlcNAc, Gal.beta.3GlcNAc, and alfa for
Gal.beta.3GalNAc and R is organic residue linked glycosidically to
the saccharide, and preferably alkyl such as method, ethyl or
propyl or ring structure such as a cyclohexyl or aromatic ring
structure optionally modified with further functional group.
[0288] Preferred monosaccharides include terminal or two or three
terminal monosaccharides of the binding epitope such as Gal,
GalNAc, GlcNAc, Man, preferably as anomeric conjugates: as
Fuc.alpha.R, Gal.beta.R, GalNAc.beta.R, GalNAc.alpha.R
GlcNAc.beta.R, Man.alpha.R. For example .alpha.3- or
.alpha.6-sialyltransferase synthesing sialyl Gal.beta.4GlcNAc is
preferably inhibited by Gal.beta.4GlcNAc or lactose. Preferred
donor analog includes CMP and derivatives for sialyltransferases
and UDP and derivatives for galactosyltransferases, the analogs
preferably interfere also with acceptor binding so that the enzyme
is released.
[0289] Sialyltransferase Catalyzed Transfer of a Natural Sialic
Acid
[0290] The invention is directed to sialyltransferase catalyzed
transfer of a natural sialic acid, preferably NeuAc, NeuGc or
Neu-O-Ac, from CMP-sialic acid to target cells. The invention
provides sialyltransferase catalyzed reaction according to
Formula
CMP-SA+target cell.fwdarw.SA-target cell+CMP,
[0291] preferably
CMP-SA+Gal.beta.4/3GlcNAc-target
cell.fwdarw.SA.alpha.3/6Gal.beta.4/3GlcNAc-target cell+CMP,
[0292] wherein SA is a sialic acid, preferably a natural sialic
acid,
[0293] preferably NeuAc, NeuGc or Neu-O--Ac and
[0294] the reaction is catalysed by a sialyltransferase enzyme
preferably by an .alpha.3-sialyltransferase and
[0295] the target cell is a cultured stem cell or stem cell or
early human blood cell (cord blood cell).
[0296] Preferred fucosyltransferase reactions synthesis of Lewis a
and Lewis x and sialylated variant thereof are:
GDP-Fuc+Gal.beta.4/3GlcNAc-target
cell.fwdarw.Gal.beta.4/3(Fuc.alpha.3/4)GlcNAc-target cell+GDP,
and/or
GDP-Fuc+SA.alpha.3Gal.beta.4/3GlcNAc-target
cell.fwdarw.SA.alpha.3Gal.beta.4/3(Fuc.alpha.3/4)GlcNAc-target
cell+GDP.
[0297] Both synthesis of sialyl-Lewis x,
SA.alpha.3Gal.beta.4(Fuc.alpha.3)GlcNAc, and sialyl-Lewis a,
SA.alpha.3Gal.beta.3(Fuc.alpha.4)GlcNAc, are preferred, sLex more
preferred, when the cells comprise mainly type 2 lacNAc acceptors
common on mesenchymal stem cells.
[0298] The reaction is catalysed by a fucosyltransferase enzyme
preferably by an .alpha.3/4-fucosyltransferase.
.alpha.4-fucosyltransferases (Fuc-TIII and -TV) are preferred for
synthesis of Lewis a. The novel fucosylated cell populations are
preferred for functional studies of the structure.
[0299] Specifically Protein Conjugated Glycosyltransferases
[0300] The present invention is directed to glycosyltransferases,
especially mammalian sialyltransferases and fucosyltransferases
which are effectively modified on lysine residues by NHS-biotin.
This is unexpected as the enzymes contain lysine residues in active
site regions, and modification of these would have been likely to
reduce substantially or destroy the enzyme activities to non-useful
levels.
[0301] The invention revealed useful but unexpectedly high reagent
amounts needed to obtain effectively biotinylated proteins. The
invention further revealed that it is possible to obtain
unexpectedly high biotinylation level and retaining useful enzyme
activity.
[0302] Preferred protein modified sialyltransferase enzymes or
other transferase according to the invention are homologous
N-acetyllactosamine sialylating .alpha.3-sialyltransferases
including ST3GalIII, ST3GalIV and ST3GalVI, most preferably
ST3GalIII-family enzymes. Preferred specifically protein modified
fucosyltransferases include .alpha.3/4-fucosyltransferases
Fuc-TIII, Fuc-TIV, Fuc-TV, Fuc-TVI, Fuc-TVII and Fuc-TIX, more
preferably Fuc-TVI and Fuc-TVII and most preferably Fuc-TVI. In a
preferred embodiment the invention is directed to biotinylated
forms of bacterial glycosyltransferases, especially
sialyltransferases and fucosyltransferases.
[0303] The invention is directed to novel substances according to
the formula
GE(-Lys)m-[NH--CO-(S)n-tag]q,
[0304] wherein GE is glycosylation modification enzyme, including
glycosyltransferring enzymes, glycocosidases and in separate
embodiment also proteases and protease inhibitors;
[0305] more preferably glycosyltransferase, more preferably
sialidase, sialyltransferase or fucosyltransferase according to the
invention,
[0306] Lys is lysine residue, presenting reactive primary amine on
side chain, alternatively an amine group may be represented by
other structure preferably N-terminus of the protein,
[0307] S is spacer, n is 0 or 1, indicating either presence or
absence of spacer, m is integer from 0 to about 20, varying between
enzymes, and indicating the average number of lysines modified, q
is integer from 1 to about 20, varying between enzymes, and
indicating the number of tag molecules conjugated.
[0308] In a preferred embodiment GE is sialyltransferase or
fucosyltransferase. In a preferred embodiment a low average level
biotinylation is performed so that m is at least 0.8, more
preferably at least 1.0, even more preferably at least 1.1, but
less than 2. Preferred lower range average biotinylation levels for
sialyltransferase are about 1.1. (preferably in range 0.8-1.4) and
about 1.6 for fucosyltransferase (preferably in range 1.3.-1.9).
The lower fucosylation is preferred especially for analysis
purposes for observing enzymes on cell surfaces, when quantitative
biotinylation of all enzyme proteins is not needed. It is realized
that the lower level biotinylated enzymes have properties closer to
the native enzyme, e.g. with regard to molecular weight.
[0309] In another preferred embodiment unexpected high
biotinylation is targeted, so that m is preferably at least 2, even
more preferably at least 2.5, even more preferably at least 3. In a
specific embodiment GE is sialyltransferase and m is between 3 and
5, more preferably between 3.2 and 4, even more preferably between
3.25 and 3.8. In a preferred embodiment GE is
.alpha.3-sialytransferase, preferably ST3GalIII and it is modified
to amine groups to contain about 3.5, more preferably about 3.4
(3.1-3.7 biotin residues). In other specific embodiment GE is
fucosyltransferase and m is between 3 and 5, more preferably
between 3.5 and 4.8 even more preferably between 3.6 and 4.5. In a
preferred embodiment .alpha.3-fucosyltransferase, preferably
Fuc-TVI is modified to amine groups to contain about 4, more
preferably about 4.3 (4.0-4.6 biotin residues).
[0310] The invention revealed that it is possible to produce highly
biotinylated sialyltransferases with useful activity. The high
biotinylation level was revealed to include range of biotin
residues so that practically each enzyme protein contains a biotin.
The highly biotinylated enzymes are especially preferred for
removal from the cell modification reactions, practically no enzyme
was left. The invention revealed extremely efficient removal of the
enzymes from reaction by solid phase conjugated binding reagent for
the tag, especially streptavidin coated magnetic particles. The
highly biotinylated active enzymes are thus preferred by the
invention.
[0311] The invention is further directed to use of biotinylated
enzymes with the reagents revealed to be effective in cell
modification such as divalent cations and/or magnesium and/or
calsium, supporting factors and non-glycoprotein. In a preferred
embodiment highly biotinylated enzyme is used in a medium
comprising biotin. The invention is especially directed to removal
of the highly biotinylated enzymes even from biotin containing
solutions, at least when the biotin concentration is not
substantially higher than that in .alpha.-MEM, and optionally 1-2
washing steps are included to remove excess biotin, before the
removal of the enzyme. In another preferred embodiment the
invention is directed to the use of the supporting factors
according to the invention and excluding biotin or using another
tag such as fluorocarbon or peptide tag not included in the
reaction mixtures.
[0312] Combined Sialyltransferase and Fucosyltransferase
Reaction
[0313] The invention is further in a preferred embodiment directed
to combined fucosyltransferase and sialyltransferase reaction with
a broad specificity, sialyltransferase sialylating
.alpha.3/4-fucosylated terminal oligosaccharide sequences such as
Lewis x and/or Lewis a, preferably at least Lewis x, and an
.alpha.3/4-fucosyltransferase reacting with both sialylated and
non-sialylated acceptors.
[0314] Combined sialyltransferase and fucosyltransferase reactions
for synthesis of sialyl-Lewis x and/or sialyl-Lewis a
Gal.beta.4/3GlcNAc (Sialyltransferase and
fucosyltransferase).fwdarw.NeuNAc.alpha.3Gal.beta.4/3(Fuc.alpha.3/4)GlcNA-
c.
[0315] More preferably synthesis of sLex
Gal.beta.4GlcNAc (Sialyltransferase and
fucosyltransferase).fwdarw.NeuNAc.alpha.3Gal.beta.4(Fuc.alpha.3)GlcNAc.
[0316] In a preferred embodiment broad specificity microbial enzyme
is used for sialylation of Lewis x and optionally also LacNAcs on
cell surface
Gal.beta.4(Fuc.alpha.3)GlcNAc (broad specificity
Sialyltransferase).fwdarw.NeuNAc.alpha.3Gal.beta.4(Fuc.alpha.3)GlcNAc
[0317] It is realized that these reactions are more effective than
reactions with mammalian transferases, because the fucosylated
sequences are not effective acceptors for mammalian
sialyltransferases.
[0318] Conjugated Enzymes
[0319] The present invention is directed to the use of the specific
enzyme for or in context of modification of the stem cells wherein
the enzyme is covalently conjugated to a tag. The conjugation
according to the invention may be performed non-specifically, e.g.
by biotinylation one or several of multiple amines on the cell
surface, or specifically.
[0320] Specific Conjugation
[0321] The specific conjugation aims for conjugation from protein
regions, which does not disturb the binding of the binding site of
the enzyme to its ligand glycan and/or donor nucleotide binding
site of a glycosyltransferase to be modified on the cell surface
glycans of stem cells according to the invention.
[0322] Preferred specific conjugation methods include chemical
conjugation from specific amino acid residues from the surface of
the enzyme protein/peptide. In a preferred method specific amino
acid residue such as cysteine is cloned to the site of conjugation
and the conjugation is performed from the cysteine. In another
preferred method N-terminal cysteine is oxidized by periodic acid
and conjugated to aldehyde reactive reagents such as
amino-oxy-methyl hydroxylamine or hydrazine structures, further
preferred chemistries include "Click" chemistry marketed by
Invitrogen and amino acid specific coupling reagents marketed by
Pierce and Molecular probes.
[0323] A preferred specific conjugation occurs from protein linked
carbohydrate such as O- or N-glycan of the enzyme, preferably when
the glycan is not close to the binding site of enzyme substrates or
longer spacer is used.
[0324] Glycan Conjugated Enzyme Protein
[0325] Preferred glycan conjugation occurs through a reactive
chemoselective ligation group R1 of the glycans, wherein the
chemical group can be specifically conjugated to second
chemoselective ligation group R2 without major or binding
destructive changes to the protein part of the enzyme.
Chemoselective ligation groups reacting with aldehydes and/or
ketones include as amino-oxy- methyl hydroxylamine or hydrazine
structures. A preferred R1-group is a carbonyl such as an aldehyde
or a ketone chemically synthesized on the surface of the protein.
Other preferred chemoselective groups include maleimide and thiol;
and "Click"-reagents (marketed by Invitrogen) including azide and
reactive group to it.
[0326] Preferred synthesis steps include
[0327] chemical oxidation by carbohydrate selectively oxidizing
chemical, preferably by periodic acid or
[0328] enzymatic oxidation by non-reducing end terminal
monosaccharide oxidizing enzyme such as galactose oxidase or by
transferring a modified aldehyde or ketone group comprising
monosaccharide residue (such as Gal Comprising CH.sub.3COCH.sub.2--
instead of OH on position 2) to the terminal monosaccharide of the
glycan.
[0329] Use of oxidative enzymes or periodic acid are known in the
art having been described in patent application directed
conjugating HES-polysaccharide to recombinant protein by
Kabi-Frensenius (WO2005EP02637, WO2004EP08821, WO2004EP08820,
WO2003EP08829, WO2003EP08858, WO2005092391, WO2005014024 included
fully as reference) and a German research institute.
[0330] Preferred methods for the transferring the terminal
monosaccharide reside includes use of mutant galactosyltransferase
as described in patent application by part of the inventors
US2005014718 (included fully as reference) or by Qasba and
Ramakrishman and colleagues US2007258986 (included fully as
reference) or by using method described in glycopegylation
patenting of Neose (US2004132640, included fully as reference).
[0331] Conjugates Including High Specificity Chemical Tag
[0332] In a preferred embodiment the enzyme is, specifically or
non-specifically conjugated to a tag, referred as T, specifically
recognizable by a ligand L, examples of tag includes such as biotin
binding ligand (strept)avidin or a fluorocarbonyl binding to
another fluorocarbonyl or peptide/antigen and specific antibody for
the peptide/antigen
[0333] Tag-Conjugate Structures
[0334] The preferred conjugate structures are according to the
Formula CONT
B-(G-)mR1-R2-(S1-)nT,
[0335] Wherein B is the enzyme, G is glycan (when the enzyme is
glycan conjugated), R1 and R2 are chemoselective ligation groups, T
is a tag, preferably biotin; S1 is an optional spacer group,
preferably C1-C10 alkyls,
[0336] m and n are integers either 0 or 1, independently.
[0337] Methods to chemically attach spacer structures ligation
groups or ligand such as (strept)avidin to solid phases is known in
the art.
[0338] Complex Structure
[0339] When the enzyme is removed by using the tag following
complex structure is preferably formed according to Formula
COMP
B-(G-)mR1-R2-(S1-)nT-L-(S2)s-SOL,
[0340] Wherein B is the enzyme, SOL is solid phase or affinity
matrix or polymer or other matrix useful for removal of the enzyme,
G is glycan (when the enzyme is glycan conjugated), R1 and R2 are
chemoselective ligation groups, T is tag, preferably biotin, L is
specifically binding ligand for the tag; S1 and S2 are optional
spacer groups, preferably C1-C10 alkyls, m, n, and s are integers
being either 0 or 1, independently and linkage between T-L can be
non-covalent high affinity binding.
[0341] Methods to chemically attach spacer structures or ligand
such as (strept)avidin to solid phases are known in the art.
[0342] Use of the Tag Conjugates
[0343] A preferred method of the tag conjugate involves following
steps:
[0344] 1) Incubating the tagged enzyme with cells
[0345] 2) Optional addition of enzyme inhibitor for the release of
the enzyme from the cells
[0346] 3) Contacting the releases tagged enzyme with a matrix
comprising the specific ligand for the tag
[0347] 4) Isolating the enzyme-matrix complex from the cells.
[0348] The matrix comprising the ligand may be solid phase or
affinity matrix or polymer or other matrix useful for removal of
the enzyme. The matrix may be used in form of magnetic particles,
column, surface of tubing or vessel, soluble or insoluble
preferably water miscible polymer.
[0349] In yet another preferred embodiment the tagged enzyme is
used together with non-tagged enzyme in order to establish the
level of non-tagged enzyme with same or very similar cell binding
properties in a cell preparation, preferably aimed for therapeutic
use, and removal of the tagged enzyme.
[0350] Novel Glycan Conjugates
[0351] The invention further revealed novel glycan conjugated
enzymes. It is realized that the glycan conjugation surprisingly
maintained the enzyme activity and allows effective reactions. The
invention is further directed multiply Tag conjugated glycans
preferably being on average about 3-15 tags, more preferably 4-12,
most preferably 4-10 tags per enzyme.
[0352] The invention is especially directed to multiply conjugated
tags on enzymes and/inhibitors according to the invention according
to the Formula
B-(G-)m[R1-R2-(S1-)nT]q Formula CONJm
[0353] Wherein B is the enzyme, SOL is solid phase or affinity
matrix or polymer or other matrix useful for removal of the enzyme,
G is glycan (when the enzyme is glycan conjugated), R1 and R2 are
chemoselective ligation groups, T is tag, preferably biotin, L is
specifically binding ligand for the tag; S1 is an optional spacer
group, preferably C1-C10 alkyls, m is integer from 1-50, indicating
number of glycan epitopes to be modified, n is integer being either
0 or 1, independently, q is integer from 1 to about 20, varying
between enzymes, and indicating the number of tag molecules
conjugated.
[0354] Methods to chemically attach spacer structures ligation
groups or ligand such as (strept)avidin to solid phases are known
in the art.
[0355] The invention is further directed to multivalent conjugates.
When the enzyme is removed by using the tag following complex
structure is preferably formed according to Formula COMPm
B-(G-)m[R1-R2-(S1-)nT-L-(S2)s-SOL]q,
[0356] q is integer from 1 to about 20, varying between enzymes,
and indicating the number of tag molecules conjugated,
[0357] other variables are as described for Formula COMP.
[0358] Novel Microbial Enzymes
[0359] The preferred bacterial enzymes includes
.alpha.2,3-sialyltransferase from Photobacterium phosphorium and
.alpha.2,6-sialyltransferase from Photobacterium damselae.
[0360] In a preferred embodiment the invention is directed to use
of sialyltransferase which can sialylate (i) both fucosylated and
non-fucosylated type II N-acetyllactosamines Gal.beta.4GlcNAc and
Gal.beta.4(Fuc.alpha.3)GlcNAc and (ii) optionally terminal type I
N-acetyl-lactosamines Gal.beta.3GlcNAc and
Gal.beta.3(Fuc.alpha.3)GlcNAc and (iii) even more preferably also
Gal.beta.3GalNAca sequences in O-glycans and/or
Gal.beta.3GalNAc.beta. in glycolipids.
[0361] Bacterial enzyme form .alpha.2,3-sialyltransferase from
Photobacterium sialylates both type 1 and type II
N-acetyllactosamines. A preferred sialyltransferase for sialylation
for all the preferred acceptor types including type I and type II
lactosamines, and glycolipid Gal.beta.3GalNAc.beta. and fucosylated
acceptors Gal.beta.4(Fuc.alpha.3)GlcNAc is a viral
sialyltransferase from myxoma virus v-ST3GalI (Sujino et al.
Glycobiology 2000 10 (3) 313-20). It is realized that the broad
specificity sialyltransferases sialylate cells most effectively to
multiple acceptor sites.
[0362] Synthesis and Analysis of Novel Sulfated Epitopes
[0363] Analysis of glycan structures of mesenchymal stem cells
revealed presence of sulfated type II N-acetyllactosamine epitopes
with sulfate on galactose residue and/or GlcNAc residue,
PCT/FI2008/050019. The applicants further had O-glycan structures
analyzed, and the positions of the sulfate modification in sulfated
N-acetyllactosamines was determined by specific galactosidases
either cleaving galactose from Gal.beta.4(SE6)GlcNAc or not
cleaving it. The analysis revealed that the sulfated
N-acetyllactosamines of human mesenchymal cells, preferably
mesenchymal stem cells contain sulfate on both Gal and GlcNAc
residues of the N-acetyllactosamines (LacNAcs).
[0364] It is realized that the sulfated N-acetyllactosamines and
their fucosylated and/or sialylated derivatives are useful markers
for the analysis of glycomodified cells.
[0365] Preferred reactions with (SE6)GlcNAc containing structures
includes fucosylation, sialylation or fucosylation and sialylation
to synthesize
[0366] a) 6-sulfo-sialyl-LacNAc, b) 6-sulfo-Lewis x and c)
6-sulfo-sialyl-Lewis x: according to formulas:
a) Gal.beta.4(SE6)GlcNAc.beta.-R
(Sialyltransferase).fwdarw.NeuNAc.alpha.3Gal.beta.4(SE6)GlcNAc.beta.-R
b) Gal.beta.4(SE6)GlcNAc.beta.-R
(fucosyltransferase).fwdarw.Gal.beta.4(Fuc.alpha.3)(SE6)GlcNAc.beta.-R,
c) Gal.beta.4(SE6)GlcNAc.beta.-R (Sialyltransferase and
fucosyltransferase).fwdarw.NeuNAc.alpha.3Gal.beta.4(Fuc.alpha.3)(SE6)GlcN-
Ac.beta.-R,
[0367] wherein R is reducing end part of the glycan preferably
2Man.alpha.3/6-linked to N-glycan core or an O-glycan structure,
preferarably 6(Gal)GalNAc.alpha.Ser/Thr.
[0368] Preferred reactions with (SE3)Gal containing structures
include fucosylation,
[0369] a) 3'sulfo-fucosyl-LacNAc according to the reaction
formula:
a)
(SE3)Gal.beta.4GlcNAc.beta.-(fucosyltransferase).fwdarw.(SE3)Gal.beta-
.4(Fuc.alpha.3)GlcNAc.beta.-R,
[0370] wherein R is reducing end part of the glycan preferably
2Man.alpha.3/6-linked to N-glycan core or an O-glycan structure,
preferably 6(Gal)GalNAc.alpha.Ser/Thr.
[0371] Preferred reactions with (SE3)Gal.beta.4(SE6)GlcNAc
containing structures includes fucosylation,
[0372] a) 3'-,6-sulfo-fucosyl-LacNAc according to the reaction:
a) (SE3)Gal.beta.4(SE6)GlcNAc.beta.-R
(fucosyltransferase).fwdarw.(SE3)Gal.beta.4(Fuc.alpha.3)(SE6)GlcNAc.beta.-
-R,
[0373] wherein R is reducing end part of the glycan preferably
2Man.alpha.3/6-linked to N-glycan core or an O-glycan structure,
preferarably 6(Gal)GalNAc.alpha.Ser/Thr.
[0374] The invention is further directed to spontaneous
fucosylation reactions to synthesize sialylated and/or fucosylated
sulfo-N-acetyllactosamines.
[0375] Preferred reagents for the recognition of acceptor
structures, preferably on non-modified cells according to the
invention include sulfo-lactosamine structure recognizing
antibodies, preferably 3' sulfo-LacNAc and 6-sulfo LacNAc,
3'-,6-sulfo-LacNAc specific antibodies and for the recognition of
product structures antibodies for the corresponding fucosylated
and/or sialylated structures. Examples of antibodies are in Table
3, in case specific antibody is not available combinations of the
antibodies can be used or combinations of antibody and glycosidase
analysis.
[0376] Effective Desialylation Reactions, and Reaction with Unusual
Specificity and Novel Cell Products
[0377] The present invention reveals an effective method to produce
highly desialyated mesenchymal stem cells. It is realized that
there are different sialylated structures on cell surface and these
are differentially available for enzymes. .alpha.3-sialidase such
as Streptococcus sp. sialidase could be used for specific
.alpha.3-desialylation. Surprisingly the inventors revealed that a
more general sialidase from Vibrio cholerae is capable of removing
effectively and selectively sialic acid residues recognized by SNA
(Sambucus nigra agglutintin) lectin, which correspond to
.alpha.6-linked sialic acids. The invention is directed to use and
method including .alpha.6-linkage favoring sialidase, preferably
Vibrio cholerae type sialidase(neuraminidase) to remove with
reasonable selectivity .alpha.6-linked sialic acids from cell
surface to obtain novel preferred cell population with
substantially reduced amount of .alpha.6-linked sialic aicds. It
was also revealed that the enzyme has some preference for sialic
aid(s) on CHO131-antibody epitope but the .alpha.6-sialic acid is
most effectively removed.
[0378] In a preferred embodiment the invention is directed to
sialylation alteration method wherein mojor part on first silic
acid structure is removed from cell surface and substantial amount
of second sialylglycan is added to the cell surface. In a preferred
embodiment .alpha.6-linked sialic acid is removed by at least 10%,
or other preferred amount according to the invention and sialic
acid with .alpha.3- or .alpha.8-linkage, preferably .alpha.3-linked
sialic acid is added preferably by a .alpha.3-sialyltransferase. In
another preferred embodiment .alpha.3-linked sialic acid is removed
by at least 10%, or other preferred amount according to the
invention and sialic acid with .alpha.6- or .alpha.8-linkage,
preferably .alpha.6-linked sialic acid is added preferably by a
.alpha.6-sialyltransferase such as ST6Gal1.
[0379] The invention is especially directed to effective reduction
of a sialylated epitope selected from a group .alpha.3-linked
sialic acid, and/or .alpha.6-linked sialic acid and/or.quadrature.
.alpha.-linked sialic acid, more preferably .alpha.3- and/or
.alpha.6-linked on a cell surface, preferably on a human
mesenchymal cell surface. The amount of sialic acid or
.alpha.3-linked sialic acid detectable by .alpha.3-sialic acid
binding lectin or antibody, preferably binding reagent selected
from the froup lectin MAA (Maackia amurensis agglutinin), MAL1 and
a sialyl-lewis x recognizing antibody, preferably CHO131.
[0380] The specific sialic acid structure is removed preferably by
at least 5% more preferably at least by 7.5%, even more preferably
at least by about 9%, even more preferably by at least by 15%, even
more preferably at least 20%. In a preferred embodiment
.alpha.6-linked sialic acid is removed by at least 10%, more
preferably by 20%, even more preferably by 30%, even more
preferably by 40%, even more preferably by 50%, even more
preferably by 60% , and most preferably at least by 70%. The
invention is further directed to novel cell populations especially
stem cells, more preferably mesenchymal stem cells comprising the
reduced sialic acid amounts. In a preferred embodiment the
invention is directed to cells with more than 35% (or other
preferred amount described above), more preferably at least about
50%, and most preferably at least about 60% reduced
.alpha.6-sialylation and optionally about 5-30% reduced
.alpha.3-sialylation. In a preferred embodiment the sialylation is
altered and the novel cell population is produced by method(s)
according to the invention including use of specifically selected
enzymes, in a preferred embodiment V. cholerae sialidase or a
sialidase with similar specificity and in another preferred
embodiment by sialidase with similar protein structure and/or by
including present cell culture reagents such as calcium and/or
magnesium 2+ cation and a non-glycoprotein, and preferably further
containing other molecules of invention supporting the cells.
[0381] In a preferred embodiment the sialic acid amount reduction
to be observed in analysis of the novel cells and/or characterizing
the novel cell product with speficic binder (antibody/lectin)
reagents is within + or -5% units of the values for MAA and/or Mal1
lectins and within + or -10% units for CHO antibody and within + or
-25% units more preferably with 20% units of the sialic acid
reduction values measured by lectin SNA as indicated in examples
and preferably the cell is human mesenchymal cells.
[0382] The invention is in general directed to novel cell
preparations according to the invention herein the modification
levels measures by specific binder reagents (lectin or antibody) or
mass spectrometry is within + or -50%, more preferably with + or
-35, even more preferably within + or -25% of the given value (e.g.
with 50% the range would be 5-15% for value 10% for e.g. value cell
portion obtainable by fax or change of sialylation measured by
alteration of glycan sialylation by mass spectrometry, preferably
N-glycans sialylation).
[0383] The invention is directed also to the preferred enzyme
reaction conditions selected from a group a buffer with similar pH,
preferably with 1.5, more preferably with 1 pH unit, and similar
salt concentrations and enzyme amounts (as Units defined in example
and/or producer of the specific enzyme) within + or -75%, + or
-50%, more preferably with + or -35, even more preferably within +
or -25% of the given value. It is further realized that enzymes can
be substituted by enzymes with higher specific activity. It is
further realized that reaction can be optimized outside these
ranges to obtain higher glycosylation modification levels,
considering the viability of the cells, and enzyme amount can be
addad to 2, 3 or 5 fold, preferably using purified enzyme
preparations and preferably removing the enzymes after reaction as
defined in invention and copending application of applicants, e.g
PCT/FI2008/050015.
EXAMPLES
Example 1
Reaction Composition for Glycan Modification of Adherent Cells in
the Presence of Divalent Cations
[0384] Materials and Methods
[0385] Cell detachment before enzymatic reactions: Bone
marrow-derived mesenchymal stem cells (BM-MSC) in 70% confluency
were detached with 0.25% trypsin/1 mM EDTA in
Ca.sup.2+/Mg.sup.2+-free PBS (Invitrogen) or 0.05% trypsin/0.5 mM
EDTA in Ca.sup.2+/Mg.sup.2+-free HBSS for 3 minutes. The
trypsinization was inhibited by adding excess of .alpha.-MEM
supplemented with 10% human serum albumin (HSA) (Albumin SPR,
Sanquin, the Netherlands). Viability of the detached BM-MSCs was
studied by Trypan blue exclusion. Immunophenotypic markers for MSC,
binding of conjugated lectins and glycoform-specific antibodies for
Lex and sLex glycan epitopes were analyzed by flow cytometry
directly after trypsinization. The detached cells were centrifuged
300.times.g for 5 min, the supernatant was completely removed and
1.times.106 cells were resuspended in 300 .mu.l enzyme reaction
buffer composed of Minimum Essential Medium (MEM) .alpha. medium
supplemented with 0.5% HSA (reaction buffer control).
[0386] Enzymatic reactions: Glycosylation enzymes and sugar donors
were added into the reaction buffer (Minimum Essential Medium (MEM)
.alpha. medium supplemented with 0.5% HSA) in the following
concentrations: 50 or 100 mU rat recombinant (Spodoptera
frugiperda) .alpha.2,3-(N)-Sialyltransferase III (SAT) (Calbiochem)
and 1 mg CMP-NeuAc, 15 or 30 mU human recombinant (Spodoptera
frugiperda) .alpha.1,3-Fucosyltransferase VI (FUT) (Calbiochem)+1
mg GDP-fucose, both SAT and FUT or 30 or 60 mU Sialidase
(Neuraminidase) C. perfringens (.alpha.2,3-6 specific). The
original enzyme buffers were exchanged to the above mentioned
reaction buffer or PBS (for Sialidase C. perfringens) by dialysis.
The BM-MSC cell suspensions were incubated in 24-well cell culture
vessels for maximally 2 hours in +37.degree. C. cell incubator,
except in the double SAT+FUT reactions, wherein FUT and GDP-Fucose
were added after one hour prior incubation with SAT. To prevent
cells to re-adhere to each others or surface of culture vessels,
the reactions were resuspended by mechanical pipetting using ART
1000E filter tips (Molecular BioProducts) with M1000 micropipette
(Biohit) every 20 minutes during the incubation. Parallel reactions
with cells only in the reaction buffer without the enzymes were
always included in each experiment. The reactions were collected
after 2 hours and cell viability and the number of cells were
determined with trypan blue exclusion.
[0387] FACS analysis: For immunophenotypic characterization of MSC
by flow cytometry (FACSAria, Becton Dickinson), antibodies against
the following molecules were used (table x): CD90, CD73, CD105,
HLA-ABC, CD34, CD45, CD14, CD19 and HLA-DR. Cell surface lectin
binding profile of the BM-MSCs was studied with FITC- or
biotin-conjugated MAA, SNA, RCA and ECA lectins before and after
the enzymatic modifications of cell glycans. FITC-conjugated
anti-streptavidin secondary antibody staining was done when
biotin-conjugated lectins were used. The following Alexa
488-conjugated anti- Lex/sLex glycoform antibodies were also used
for FACS analysis: CD15 (TG-1) Lex, PSGL-1 (core 2 O-glycan) sLex
and sCD15 (sLex). 1.times.10.sup.5 BM-MSCs were labelled in
Ca.sup.2+-free PBS supplemented with 2 mM EDTA and 1% BSA. Analysis
was performed using the FACSDiva software (Beckton Dickinson).
[0388] Results
[0389] BM-MSC viability and immunophenotypic markers after in vitro
glycomodification Viability of the BM-MSCs never fell below 98% and
their cell surface expression of CD90, CD73, CD 105, HLA-ABC, CD34,
CD45, CD 14, CD19 and HLA-DR remained unaltered after the
.alpha.2,3-sialylation (SAT) or .alpha.1,3-fucosylation (FUT)
reactions carried out in the reaction conditions described above
(data not shown).
[0390] Effects of Glycomodifications on BM-MSC Cell Surface Binding
of Plant Lectins and Lex and sLex Identifying Antibodies
[0391] The glycomodifications were validated by FACS analysis of
the binding of plant lectins and glycoform specific antibodies
(Table 1) onto the cell surface. The results are summarized in FIG.
1.
[0392] A small increase in the percentage of MAA high-binding cells
was noted after 2 h 50 mU .alpha.2,3-SAT reactions in the divalent
cation containing buffer. Simultaenously, the RCAPE population
staining was reduced with similar percentage values. The use of a
higher amount of the enzyme, 100 mU for 2 h, for the sialylation
produced a more robust increase in the MAA binding.
[0393] It is noteworthy that an increase in cell surface
sialylation (both .alpha.2,3- and 6 linked sialic acids) directly
after 0.25% trypsinization could be produced merely with the
reaction buffer composition as compared to the control cells (FIG.
1). This was demonstrated by the MAA and SNA stainings, which were
comparable to the decrease in the RCAPE staining. An increase in
the staining intensity after incubating BM-MSC in the reaction
buffer as compared to the control cells could also be observed for
the core-2 sLex antibody, but not for the sLex/CSLEX1, or the CD90,
CD105 and CD73 antibodies. The .alpha.1,3-fucosylation reaction was
very efficient and >90% of the cells expressed the sLex/CSLEX1
epitope in the reaction composition and time chosen (FIG. 1,
sLex/CSLEX1 antibody). Also the core-2 sLex antibody stained
>90% of the cells after the .alpha.1,3-fucosylation reaction. A
proceeding 1 h .alpha.2,3-sialylation inhibited the fucosylation
reaction (FIG. 1).
[0394] There were no differences between the strong and mild
trypsinization protocols before the 2 h reactions with or without
enzyme concerning MAAhigh and core-2 O-glycan sLex antibody cell
surface binding (FIG. 1). The sialidase reaction reduced the
MAAhigh and SNAhigh lectin cell surface binding (FIG. 1). The
sialidase reaction, however, also reduced ECA binding. Incubating
the cells in the reaction buffer and excess NeuAc increased MAAhigh
and especially SNAhigh binding (FIG. 1).
Example 2
Effect of Divalent Cations and Shear Stress to Aggregation and
Morphology of Bone Marrow Mesenchymal Stem Cells (BM-MSCs)
[0395] Materials and Methods
[0396] BM-MSCs in 70% confluency were detached with 0.25% trypsin/1
mM EDTA in Ca.sup.2+/Mg.sup.2+-free PBS (Invitrogen) for 3 minutes.
Trypsinization was inhibited by adding excess .alpha.-MEM+10% human
serum albumin HSA (Albumin SPR, Sanquin, the Netherlands). The cell
suspension was centrifuged, supernatant removed and
1.75.times.10.sup.6 cells were resuspended in either 300 .mu.l
Minimum Essential Medium (MEM) a medium supplemented with 0.5% HSA
or Ca.sup.2+/Mg.sup.2+-free Hanks' Balanced Salt Solution (HBSS)
supplemented with 0.1% HSA. The BM-MSC cell suspensions were
incubated in 24-well cell culture vessels for 2 hours in
+37.degree. C. cell incubator. In a part of the culture wells, the
cells were resuspended by mechanical pipetting through ART 1000E
filter tips (Molecular BioProducts) attached to M1000 micropipette
(Biohit) every 20 minutes during the incubation. Representative
phase contrast microscope pictures of cells in suspension were
taken at the beginning of the experiment and after 1 h and 2 h. The
cells in suspension were collected at the end of the experiment and
cell viability and the number of cells were determined by the
trypan blue exclusion.
[0397] Results
[0398] As shown in FIG. 2, BM-MSCs in suspension exhibited a
heterogeneous cell population with cells of different sizes
directly after trypsinization (time point 0') . After 1-2 hour
incubation a change in cell morphology could be observed between
cells in the .alpha.-MEM reaction buffer and
Ca.sup.2+/Mg.sup.2+-free HBSS. The cell suspension in
Ca.sup.2+/Mg.sup.2+-free HBSS contained more large, granular cells
and small cells with bleb-like structures on their plasma membrane.
The cells suspended in the Ca.sup.2+- and Mg.sup.2+-containing
.alpha.-MEM reaction buffer contained more cells characterized by a
round and clear, mononuclear cell-like morphology and intact plasma
membranes (in FIG. 2, compare cells marked with arrows in panels).
The cells aggregated more to each others in the .alpha.-MEM
reaction buffer, but the aggregation could be inhibited by
mechanical resuspension every 20 min. None of the reaction
conditions could completely inhibit the cells from adhering to the
cell culture vessel bottom as indicated in the total number of
cells in suspension after 2 h incubation (Table 2). The adherence
to cell culture bottom could also be inhibited by the sequential
resuspension. There were a higher number of viable cells in the
suspension phase, when the cells were incubated in the
.alpha.-MEM+0.5% HSA reaction buffer than when incubated in the
Ca.sup.2+/Mg.sup.2+-free HBSS (Table 2).
Example 3
Mild or Strong Trypsinization of BM-MSCs and the Inhibition of the
Trypsinization
[0399] Materials and Methods
[0400] BM-MSC in 70% confluency were detached with either 0.25%
trypsin/1 mM EDTA in Ca.sup.2+/Mg.sup.2+-free PBS (Invitrogen) or
0.05% trypsin/0.5 mM EDTA in Ca.sup.2+/Mg.sup.2+-free HBSS for 3
minutes. Trypsinization was inhibited by adding excess of either
.alpha.-MEM+10% fetal calf serum (FCS) (Invitrogen) or
.alpha.-MEM+10% human serum albumin (HSA) (Albumin SPR, Sanquin,
the Netherlands). Cell viability and the number of cells were
determined with trypan blue exclusion and representative phase
contrast microscope pictures were taken with 10.times.
objective.
[0401] Results
[0402] Both mild (0.05% trypsin) and 5.times. stronger (0.25%
trypsin) trypsinization detached the cells into the unicellular
cell suspension. Their viability in both cases were .gtoreq.98%.
Equally viable cells could be produced if trypsinization was
inhibited by adding 10% HSA or 10% FCS (FIG. 3). Human serum
albumin can thus be used as a xenoantigen-free inhibitor of
trypsinization.
Example 4
Glycan Controlled Enzyme
[0403] Glycosylation of commercial sialyl- or fucosyltransferase
(Calbiochem CA) enzyme produced in insect cells is controlled by
releasing the glycans, purifying the glycans and MALDI-TOF mass
spectrometry (WO publication by the inventors, filed Jul. 11,
2005). Potentially allergenic insect type glycans are observed and
released by exoglycosidase enzymes as described for the insect
glycans such as .alpha.-mannosidase, .beta.-mannosidase .alpha.3-
or .alpha.3/.alpha.6-fucosidases and hexosaminidase (such as Jack
bean hexosaminidase), (WO publication by the inventors, filed Jul.
11, 2005).
[0404] Glycosyltransferase-derived glycan structures. We detected
that glycosylated glycosyltransferase enzymes can contaminate cells
in modification reactions. For example, when cells were incubated
with recombinant fucosyltransferase or sialyltransferase enzymes
produced in S. frugiperda cells, N-glycosidase and mass
spectrometric analysis of cellular and/or cell-associated
glycoproteins resulted in detection of an abundant neutral N-glycan
signal at m/z 1079, corresponding to [M.sup.+Na].sup.+ ion of
Hex3HexNAc2dHex1 glycan component (calc. m/z 1079.38). Typically,
in recombinant glycosyltransferase treated cells, this glycan
signal was more abundant than or at least comparable to the cells'
own glycan signals, indicating that insect-derived glycoconjugates
are a very potent contaminant associated with recombinant
glycan-modified enzymes produced in insect cells. Moreover, this
glycan contamination persisted even after washing of the cells,
indicating that the insect-type glycoconjugate corresponding to or
associated with the glycosyltransferase enzymes has affinity
towards cells or has tendency to resist washing from cells. To
confirm the origin of the glycan signal, we analyzed glycan
contents of commercial recombinant fucosyltransferase and
sialyltransferase enzyme preparations and found that the m/z 1079
glycan signal was a major N-glycan signal associated with these
enzymes. Corresponding N-glycan structures, e.g.
Man.alpha.3(Man.alpha.6)Man.beta.4GlcNAc(Fuc.alpha.3/6)GlcNAc(.beta.-N-As-
n), have been described previously from glycoproteins produced in
S. frugiperda cells (Staudacher et al., 1992; Kretzchmar et al.,
1994; Kubelka et al., 1994; Altmann et al., 1999). As described in
the literature, these glycan structures, as well as other glycan
structures potentially contaminating cells treated with recombinant
or purified enzymes, especially insect-derived products, are
potentially immunogenic in humans and/or otherwise harmful to the
use of the modified cells. It is concluded that glycan-modifying
enzymes must be carefully selected for modification of human cells,
especially for clinical use, not to contain immunogenic glycan
epitopes, non-human glycan structures, and/or other glycan
structures potentially having unwanted biological effects or in a
preferred embodiment the glycan structures are removed or degraded
to non-harmful ones.
Example 5
Enzymes and Conjugations
[0405] Conjugated Sialyltransferase and Fucosyltransferase
Enzymes
[0406] The SAT and FUT enzymes were recombinant rat
.alpha.2,3-N-sialyltransferase and recombinant
.alpha.1,3-fucosyltransferase VI, respectively, expressed in S.
frugiperda insect cells (Calbiochem).
[0407] Conjugation to Protein
[0408] The enzymes were biotinylated with
N-hydroxysuccinimide-activated sulfo-biotin with aminocaproate
spacer (sulfo-NHS-LC-biotin; Pierce). In the reaction, 5 .mu.g of
enzyme was incubated with either 0.65 nmol (about 5 times molar
excess) or 6.5 nmol (about 50 times molar excess) biotinylation
reagent in 50 .mu.l of 50 mM HEPES buffer pH 8 at +4.degree. C. for
2 hours. The reactions were stopped by adding a molar excess of
Tris-HCl buffer pH 7.5.
[0409] Conjugation to Glycan
[0410] For periodate oxidation, the commercial glycerol-containing
enzyme preparates were transferred into phosphate buffered saline
solution with gel filtration chromatography in NAP-5 columns
(Amersham-Pharmacia/GE Healthcare). Periodate oxidation of glycan
chains was performed for 5 .mu.g of either SAT or FUT in 40 .mu.l
of phosphate buffered saline (PBS) containing 8 mM NaIO.sub.4, at
+4.degree. C. in the dark for 2-3 hours. Biotin conjugation to
oxidized glycan chains was performed by adding 6.5 nmol (about 100
times excess) of biotin-amidohexanoic acid hydrazide (BAAH; Pierce)
to the glycan-oxidized enzymes and incubating at +4.degree. C. for
2-16 hours. The enzymes were isolated for subsequent use.
[0411] Activity Assays
[0412] Activity of conjugated enzymes was measured by performing
standard reactions and comparing the amount of sialylated products
to non-conjugated enzyme.
[0413] In a typical radiochemical SAT assay, 0.4 mU/ul enzyme was
incubated with 1 mM CMP-[C-14]Neu5Ac and 0.5 mM acceptor
glycoprotein (asialofetuin from Sigma-Aldrich) in 50 mM MOPS buffer
pH 7.4 at +37.degree. C. Aliquots from various time points were
collected. Protein fraction was separated from the donor substrate
by reversed phase solid-phase extraction and measured by liquid
scintillation counting according to standard procedures. The
amounts of radioactivity transferred to protein were compared. FUT
activity was measured similarily using 0.04 mU/.mu.l FUT,
radiolabeled GDP-Fuc donor, and additionally 20 mM MnCl.sub.2.
[0414] In a typical mass spectrometric assay, 0.2 mU/.mu.l enzyme
was incubated with 8 mM CMP-Neu5Ac and 4 mM LacNAc acceptor glycan
(lacto-N-neotetraose) in 50 mM MOPS buffer pH 7.4 at +37.degree.
C., optionally containing 0.2 mg/ml albumin. Aliquots from various
time points were collected. FUT activity was measured similarily
using 0.05 mU/.mu.l FUT, 8 mM GDP-Fuc donor, 4 mM acceptor,
optionally with 20 mM MnCl2, and preferentially without albumin.
The reaction level was analyzed by MALDI-TOF mass spectrometry
using standard procedures, and relative signal intensities
(correlating with corresponding relative molar amounts in the
sample) of acceptor and product glycans were compared.
[0415] Enzymes in Cell-Optimized Liquid Compositions
[0416] SAT and FUT were optionally changed into cell-optimized
solutions, preferentially .alpha.-MEM (Gibco/Invitrogen) and
optionally using filtration concentration or dialysis. Optionally,
human serum albumin (HSA; Finnish Red Cross Blood Service) was
added, most commonly to concentration of 0.56% (5.6 mg/ml).
[0417] Conjugation Level Analysis
[0418] Conjugation level was analyzed by comparing molecular mass
of non-conjugated and conjugated proteins in MALDI-TOF mass
spectrometry using standard procedures.
[0419] Removal of Conjugated Enzyme
[0420] Biotinylated enzymes were removed from solution by
contacting them with affinity matrix, preferentially with
streptavidin-coated magnetic particles (Dynal). Removal of enzyme
was visualized by SDS-PAGE and blotting with
streptavidin-peroxidase conjugate and color reaction according to
standard procedures.
[0421] Results
[0422] Protein-conjugated SAT and FUT, 5 times molar excess of
biotinylation reagent:
[0423] SAT: on average 1.1 biotin unit was conjugated (+360 Da mass
increase observed in MALDI-TOF MS) and comparable activity was
retained as measured by the mass spectrometric assay;
[0424] FUT: on average 1.6 biotin unit was conjugated (+540 Da mass
increase observed in MALDI-TOF MS) and comparable activity was
retained as measured by the mass spectrometric assay.
[0425] Protein-conjugated SAT and FUT, 50 times molar excess of
biotinylation reagent:
[0426] SAT: on average 3.4 biotin units were conjugated (+1170 Da
mass increase observed in MALDI-TOF MS) and comparable activity was
retained as measured by the mass spectrometric assay;
[0427] FUT: on average 4.3 biotin units were conjugated (+1450 Da
mass increase observed in MALDI-TOF MS) and comparable activity was
retained as measured by the mass spectrometric assay.
[0428] Glycan-Conjugated Glycosyltransferases:
[0429] The glycan-conjugated SAT showed similar activity as
non-conjugated SAT when analyzed by the mass spectrometric assay.
Mass spectrometric analysis of glycan conjugated sialyltransferase
indicated high conjugation to glycans being about 5-10 biotins per
enzyme.
[0430] Removal of Conjugated Enzymes from Solution
[0431] The biotin-conjugated proteins could be readily removed from
solution by streptavidin-coated magnetic particles, as visualized
for protein-biotin-conjugated SAT in FIG. 5. The results
demonstrated that no enzyme could be detected in solution after the
affinity-adsorption step, translating into nearly 100% efficiency,
preferentially over 95% efficiency, and more preferentially over
99% efficiency in enzyme removal (FIG. 5).
[0432] Cell-Optimized Liquid Compositions
[0433] The SAT and FUT enzyme preparations in .alpha.-MEM and in
.alpha.-MEM supplemented with HSA (0.56%) were active as measured
by the mass spectrometric assay. Further, they showed good
stability. For example, the HSA-containing SAT preparation in
.alpha.-MEM was stable at +4.degree. C. for over 16 days (in which
time over 75% of original activity was retained as measured by the
mass spectrometric assay). Further, FUT in HSA-containing
.alpha.-MEM was active without adding Mn.sup.2+ or other divalent
cations to the standard .alpha.-MEM.
[0434] Bacterial Sialyltransferases in Cell Modification
[0435] The SAT enzymes were recombinant
.alpha.2,3-sialyltransferase (JT-ISH-224 and JT-ISH-467) from
Photobacterium phosphorium and .alpha.2,6-sialyltransferase (JT160)
from Photobacterium damselae expressed in E. coli (Japan Tobacco
Inc., Shizuoka, Japan).
[0436] Sialylation of Human Type Glycan Acceptors
[0437] In a typical assay reaction, 0.2 mU/.mu.l enzyme was
incubated with 8 mM CMP-Neu5Ac and 4 mM LacNAc acceptor glycan
(lacto-N-neotetraose) in 20 mM Tris-HCl buffer pH 7.5 at
+37.degree. C., containing 0.2 mg/ml albumin and sodium chloride
concentration of 150 mM (up to 0.5M was adviced by the
manufacturer). Aliquots were collected from several time points.
The reaction level was analyzed by MALDI-TOF mass spectrometry
using standard procedures, and relative signal intensities
(correlating with corresponding relative molar amounts in the
sample) of acceptor and product glycans were compared, and the
enzymes were highly active towards the acceptor.
[0438] Novel Bacterial Enzymes in Cell-Optimized Liquid
Compositions
[0439] SATs were optionally changed into cell-optimized solutions,
preferentially .alpha.-MEM (Gibco/Invitrogen) and optionally using
filtration concentration or dialysis. Human serum albumin (HSA;
Finnish Red Cross Blood Service) was added to concentration of 0.2
mg/ml. The salt concentration was lowered to physiological salt
contained within the .alpha.-MEM. The enzyme was highly active when
analyzed by MALDI-TOF mass spectrometry with lacto-N-tetraose
acceptor as described above.
[0440] Activity Compared to Mammalian Recombinant Enzyme
[0441] The activity of the enzymes was measured relative to
equivalent nominal activities according to manufacturer's
specifications. The bacterial enzyme was over 10 times more active
than the recombinant mammal .alpha.2,3-N-sialyltransferase produced
in insect cells by Calbiochem.
[0442] Neuraminidase or sialyltransferase is biotinylated as
described in catalog of Pierce. Biotinylated neuraminidase or
sialyltransferase enzyme is incubated with cells to modify the
glycosylation of the target cells as described in the invention.
The enzyme is removed by (strept)avidin magnetic beads (e.g.
Miltenyi or Dynal) optionally with presence of neuraminic acid and
or sialyltransferase acceptor (N-acetyllactosamine or lactose).
Example 6
Modification of BM-MSC Cells
[0443] Materials and Methods
[0444] Cells: Bone marrow-derived mesenchymal stem cells (BM-MSCs)
were obtained as described by Leskela et al. (2003). After initial
culture establishment, BM-MSCs (<passage 10) were cultured in a
humidified 5% CO.sub.2 atmosphere at +37.degree. C. in Minimum
Essential Alpha-Medium (a-MEM) (Gibco) supplemented with 10% FCS,
20 mM Hepes, 10 ml/l penicillin/streptomycin and 2 mM
L-glutamine.
[0445] .alpha.2,3-Sialyltransferase enzymatic modification: Cell
culture media was collected for further analysis before the
detachment. For the enzymatic modifications, BM-MSCs at 70-80%
confluency were detached with PBS+2 mM Na-EDTA (Versene) for 30 min
at +37.degree. C. The number of detached cells were calculated in
Burker chamber and control (0-) cells were washed four times in
cold Ca.sup.2-free PBS and frozen as cell pellets at -70.degree. C.
for mass spectrometric analysis. All centrifugation steps were
performed at 300.times.g for 5 min. 1.times.10.sup.6 BM-MSCs were
suspended in 300 .mu.l reaction buffer consisting of .alpha.-MEM
and 0.5% bovine serum albumin (BSA, >=99% pure). The enzymatic
reactions were performed in 24-well cell culture plates in a
humidified 5% CO2 atmosphere at +37.degree. C. for either 2 or 4
hours. The reactions were controlled for attachment to the cell
culture dish by suspending the cells every 30 min during the
incubations. Control reactions were performed simultaneously with
cells in reaction buffer only for 2 or 4 hours. The following
enzymatic conditions were tested (i) 50 mU recombinant
.alpha.2,3-(N)-Sialyltransferase (Calbiochem) and 1 mg CMP-Neu5Ac
(donor), (ii) 10 mU (1/5) recombinant
.alpha.2,3-(N)-Sialyltransferase and 1 mg CMP-Neu5Ac, (iii) 50 mU
recombinant .alpha.2,3-(N)-Sialyltransferase and 0.2 mg (1/5)
CMP-Neu5Ac, and (iv) 50 mU inactivated (boiled for 5 min and
transferred directly to ice) recombinant
.alpha.2,3-(N)-Sialyltransferase and 1 mg CMP-Neu5Ac. The enzymatic
reactions were stopped by adding excess (2 ml) of cold
Ca.sup.2+-free PBS or Ca.sup.2+-free PBS supplemented with 75 mM
lactose to the reactions. Cell viability was determined by trypan
blue staining and microscopic analysis in a Burker chamber. The
cells were centrifuged at 300.times.g for 5 min and washing was
repeated additionally 3 times. After the last wash the cells were
divided in two and half the cells were pelleted by centrifugation
and frozen at -70.degree. C. for further mass spectrometric
N-glycan analysis and half the cells were used subsequently in flow
cytometric analysis. The MALDI-TOF mass spectrometric analysis was
performed for N-glycosidase F liberated N-glycans essentially as
described (Hemmoranta H. et al., 2007. Exp. Hematol.).
[0446] Results
[0447] Cellular glycan modification with sialyltransferase: In the
reaction, cell surface terminal N-acetyllactosamine (LN) units were
sialylated as demonstrated by N-glycan structural analyses as
follows. Reaction efficiency and level of terminal LN modification
was followed by MALDI-TOF mass spectrometric profiling of the
neutral N-glycan fraction. Three glycan signals were used as
indicators of the reaction efficiency, namely at m/z 1622
(corresponding to hybrid-type N-glycan Hex6HexNAc3 Na-adduct
signal, with documented one terminal LN unit), m/z 1663
(corresponding to complex-type N-glycan Hex5HexNAc4 Na-adduct
signal, with documented two terminal LN units), and m/z 2028
(corresponding to complex-type N-glycan Hex6HexNAc5 Na-adduct
signal, with documented three terminal LN units). These three
indicator signals were good indicators for the overall sialylation
level change. In the table below, reaction level is calculated by
the equation:
100%-100%*(I1622+I1663+I2028)a/(I1622+I1663+I2028)b
[0448] wherein Ix is relative proportion of glycan signal x (% of
total glycan profile), "a" indicates signals after the enzyme
reaction and "b" indicates those in the control reaction. The
disappearance of sialic acid from the molecules indicated an
increase of sialylation level. The glycan signal at m/z 1257
(control, corresponding to Na-adduct of Hex5HexNAc2 high-mannose
type N-glycan) stayed between 5.7%-6.8% in all conditions, showing
that modification was specific.
[0449] condition: relative amount of glycan signals (% of total
glycan profile):
TABLE-US-00001 1622 1663 2028 reaction level (%) 0-(cells in
culture) 1.33% 2.10% 0.40% 3% buffer control 1.54% 2.02% 0.40% 0%
inactivated enzyme 1.34% 1.94% 0.48% 5% 2 h reaction 0.77% 0.32%
0.00% 72% 4 h reaction 0.52% 0.32% 0.00% 79% 1:5 donor 0.89% 0.54%
0.00% 64% 1:5 enzyme 0.95% 0.67% 0.00% 59%
[0450] The results indicated that:
[0451] 1) an exogenous, functional enzyme was needed for an
efficient reaction. This was indicated by negligible levels of the
reaction with a heat-inactivated enzyme (5% reaction level), as
compared to both the control cells (3%, by reference) and buffer
control (0%).
[0452] 2) the highest effective reaction level was about 80% (79%)
in the 4 h optimized reaction conditions,
[0453] 3) a 2 h reaction time (72%) was nearly as efficient as 4 h
reaction (79%), however, a longer reaction time produced a higher
reaction level,
[0454] 4) a lower amount of either enzyme (1:5 enzyme) or donor
substrate (1:5 donor) resulted in less efficient reactions, 59% and
64%, respectively, showing that the optimized reaction conditions
were critical for an efficient removal of LN units from cells.
[0455] When the cell culture and reaction media were analyzed by
the same method, it was additionally detected that glycoprotein
components containing a LN group when added to the medium, were
efficient substrates of the enzymatic modification, therefore
competing with the cellular modification if added to the reaction
solution.
[0456] Contamination with insect derived enzyme and its removal:
m/z 1079, corresponding to sodium adduct ion of Hex3HexNAc2dHex1, a
paucimannosidic insect N-glycan/low-mannose type human N-glycan.
Below are results from mass spectrometric measurements of the
relative amounts of m/z 1079 glycan signal in different
reaction/wash conditions.
[0457] condition: relative amount of m/z 1079 glycan signal (% of
total glycan profile):
TABLE-US-00002 Difference to Relative control % difference to 4 h
buffer control 0.75% 4 h reaction 1.45% 0.70 inactivated enzyme
1.48% 0.73 1:5 donor 1.47% 0.72 1:5 enzyme 1.09% 0.34 49% wash opt
A 0.84% 0.09 12%
[0458] The results indicated that:
[0459] 1) low level of the m/z 1079 glycan signal were present in
the cells before addition of the enzyme due to endogenous
low-mannose N-glycans (0.75% relative amount in the "buffer
control" condition),
[0460] 2) all reaction conditions with full amount of enzyme
resulted in contamination with insect-derived glycan (c. 1.5%
relative amount in the "4 h reaction", "inactivated enzyme", and
"1:5 donor" conditions),
[0461] 3) a lower amount of added enzyme resulted in a lower level
of contamination (1.1% relative amount in the "1:5 enzyme"
condition),
[0462] 4) 75 mM lactose included in the washing buffers resulted in
efficient wash of the insect-derived glycan contamination (0.84%
relative amount in the "wash opt A" condition), and
[0463] 5) incomplete reaction may increase enzyme contamination
(1.47% versus 1.45% in the "1:5 donor" and "4h reaction"
conditions, respectively).
Example 7
Production of a Tag Glyco-Conjugated Enzyme
[0464] Mammalian glycosyltransferase (e.g.
.beta.4-galactosyltransferase, bovine GalT1) is treated with
.alpha.-sialidase, and .beta.-galactosidase. Ketone modified Gal is
transferred from ketone modified Gal-UDP to the terminal
monosaccharide GlcNAc-residue by mutant galactosyltransferase as
described in patent application by part of the inventors
US2005014718 (included fully as reference) or by Qasba and
Ramakrishman and colleagues US2007258986 (included fully as
reference) or by using methods described in glycopegylation
patenting of Neose (US2004132640, included fully as reference). The
ketone is reacted with excess of amino-oxy-biotin (or
hydrazide-biotin).
Example 8
Production of Cells
[0465] Cord Blood Mesenchymal Stem Cell Lines
[0466] Collection of umbilical cord blood. Human term umbilical
cord blood (UCB) units were collected after delivery with informed
consent of the mothers and the UCB was processed within 24 hours of
the collection. The mononuclear cells (MNCs) were isolated from
each UCB unit diluting the UCB 1:1 with phosphate-buffered saline
(PBS) followed by Ficoll-Paque Plus (Amersham Biosciences, Uppsala,
Sweden) density gradient centrifugation (400 g/40 min). The
mononuclear cell fragment was collected from the gradient and
washed twice with PBS.
[0467] Umbilical cord blood cell isolation and culture.
CD45/Glycophorin A (GlyA) negative cell selection was performed
using immunolabeled magnetic beads (Miltenyi Biotec). MNCs were
incubated simultaneously with both CD45 and GlyA magnetic
microbeads for 30 minutes and negatively selected using LD columns
following the manufacturer's instructions (Miltenyi Biotec). Both
CD45/GlyA negative elution fraction and positive fraction were
collected, suspended in culture media and counted. CD45/GlyA
positive cells were plated on fibronectin (FN) coated six-well
plates at the density of 1.times.10.sup.6/cm.sup.2. CD45/GlyA
negative cells were plated on FN coated 96-well plates (Nunc) about
1.times.10.sup.4 cells/well. Most of the non-adherent cells were
removed as the medium was replaced next day. The rest of the
non-adherent cells were removed during subsequent twice weekly
medium replacements.
[0468] The cells were initially cultured in media consisting of 56%
DMEM low glucose (DMEM-LG, Gibco,) 40% MCDB-201 (Sigma-Aldrich) 2%
fetal calf serum (FCS), 1.times. penicillin-streptomycin (both from
Gibco), 1.times. ITS liquid media supplement
(insulin-transferrin-selenium), 1.times. linoleic acid-BSA,
5.times.10-8 M dexamethasone, 0.1 mM L-ascorbic acid-2-phosphate
(all three from Sigma-Aldrich. In later passages (after passage 7)
the cells were also cultured in the same proliferation medium
except the FCS concentration was increased to 10%.
[0469] Plates were screened for colonies and when the cells in the
colonies were 80-90% confluent the cells were subcultured. At the
first passages when the cell number was still low the cells were
detached with minimal amount of trypsin/EDTA (0.25%/1 mM, Gibco) at
room temperature and trypsin was inhibited with FCS. Cells were
flushed with serum free culture medium and suspended in normal
culture medium adjusting the serum concentration to 2%. The cells
were plated about 2000-3000/ cm2. In later passages the cells were
detached with trypsin/EDTA from defined area at defined time
points, counted with hematocytometer and replated at density of
2000-3000 cells/cm.sup.2.
[0470] Bone Marrow-Derived Stem Cells.
[0471] Bone marrow (BM)-derived MSCs were obtained as described by
Leskela et al. (2003). Briefly, bone marrow obtained during
orthopedic surgery was cultured in Minimum Essential Alpha-Medium
(.alpha.-MEM), supplemented with 20 mM HEPES, 10% FCS, 1.times.
penicillin-streptomycin and 2 mM L-glutamine (all from Gibco).
After a cell attachment period of 2 days the cells were washed with
Ca.sup.2- and Mg.sup.2- free PBS (Gibco), subcultured further by
plating the cells at a density of 2000-3000 cells/cm.sup.2 in the
same media and removing half of the media and replacing it with
fresh media twice a week until near confluence.
[0472] Flow Cytometric Analysis of Mesenchymal Stem Cell
Phenotype.
[0473] Both UBC and BM derived mesenchymal stem cells were
phenotyped by flow cytometry (FACSCalibur, Becton Dickinson).
Fluorescein isothicyanate (FITC) or phycoerythrin (PE) conjugated
antibodies against CD13, CD14, CD29, CD34, CD44, CD45, CD49e, CD73
and HLA-ABC (all from BD Biosciences, San Jose, Calif.), CD105
(Abcam Ltd., Cambridge, UK,) and CD133 (Miltenyi Biotec) were used
for direct labeling. Appropriate FITC- and PE-conjugated isotypic
controls (BD Biosciences) were used. Unconjugated antibodies
against CD90 and HLA-DR (both from BD Biosciences) were used for
indirect labeling. For indirect labeling FITC-conjugated goat
anti-mouse IgG antibody (Sigma-Aldrich) was used as a secondary
antibody.
[0474] The UBC derived cells were negative for the hematopoietic
markers CD34, CD45, CD14 and CD133. The cells stained positively
for the CD13 (aminopeptidase N), CD29 (.beta.1-integrin), CD44
(hyaluronate receptor), CD73 (SH3), CD90 (Thy1), CD105
(SH2/endoglin) and CD 49e. The cells stained also positively for
HLA-ABC but were negative for HLA-DR. BM-derived cells showed to
have similar phenotype. They were negative for CD 14, CD34, CD45
and HLA-DR and positive for CD 13, CD29, CD44, CD90, CD105 and
HLA-ABC.
[0475] Cell Harvesting for Glycome Analysis.
[0476] 1 ml of cell culture medium was saved for glycome analysis
and the rest of the medium removed by aspiration. Cell culture
plates were washed with PBS buffer pH 7.2. PBS was aspirated and
cells scraped and collected with 5 ml of PBS (repeated two times).
At this point small cell fraction (10 .mu.l) was taken for
cell-counting and the rest of the sample centrifuged for 5 minutes
at 400 g. The supernatant was aspirated and the pellet washed in
PBS for an additional 2 times.
[0477] The cells were collected with 1.5 ml of PBS, transferred
from 50 ml tube into 1.5 ml collection tube and centrifuged for 7
minutes at 5400 rpm. The supernatant was aspirated and washing
repeated one more time. Cell pellet was stored at -70.degree. C.
and used for glycome analysis.
[0478] Biological Use of Novel Cells
[0479] The cells can be used for in vivo imaging trials and in
animal models such as PET imagining e.g. as described in Min J J et
al. (2006) Ann Nucl Med 20,(3) 165-70 or Kang W J et al. (J Nucl
Med 47, 1295-1301).
Example 9
Modification of Adherent or Detached Stem Cells
[0480] Materials and Methods
[0481] Cells: Bone marrow-derived mesenchymal stem cells (BM-MSCs)
line 168 were obtained as described by Leskela et al. (2003). After
initial culture establishment, BM-MSCs passage 10) were cultured in
a humidified 5% CO.sub.2 atmosphere at +37.degree. C. in Minimum
Essential Alpha-Medium (.alpha.-MEM) (Invitrogen) supplemented with
10% FCS, 20 mM Hepes, 10 ml/l penicillin/streptomycin and 2 mM
L-glutamine.
[0482] .alpha.2,3-Sialyltransferase enzymatic modification: For the
enzymatic modifications, BM-MSCs at 70-80% confluency were either
detached with PBS+0.54 mM Na-EDTA (0.02% Versene) for 20 min at
+37.degree. C. or were washed once with PBS. The number of detached
cells were calculated in Burker chamber and control (sample Versene
0-) cells were washed four times in cold Ca.sup.2+-free PBS and
frozen as cell pellets at -70.degree. C. for mass spectrometric
analysis. All centrifugation steps were performed at 300.times.g
for 5 min. 300 .mu.l SAT reaction buffer consisting of .alpha.-MEM
(Invitrogen), 0.5% human serum albumin (Albumin SPR, Sanquin), 50
mU rat recombinant .alpha.2,3-(N)-Sialyltransferase (SAT,
Calbiochem) and 1 mg CMP-Neu5Ac (donor) was added to either
adherent cells in one O10 cm cell culture vessel (sample
.alpha.2,3SAT+Versene) or to detached cells in suspension from one
O10 cm cell culture vessel transferred to a 24-well cell culture
plate (sample Versene+.alpha.2,3 SAT). The reactions were performed
in a humidified 5% CO2 atmosphere at +37.degree. for 2 hours. The
cells in suspension were resuspended every 20 min during the
incubation. The enzymatic reactions were stopped by adding excess
of cold PBS. The adherent cells were subsequently detached as
described previously. Cell viability was determined by Trypan blue
exclusion and microscopic analysis in a Burker chamber for all
cells after modifications and detachment. The cells were
centrifuged and washing with PBS was repeated additionally 3 times.
The cells were finally pelleted by centrifugation and frozen at
-70.degree. C. for further mass spectrometric N-glycan analysis.
The MALDI-TOF mass spectrometric analysis was performed for
N-glycosidase F liberated N-glycans essentially as described
(Hemmoranta H. et al., 2007. Exp. Hematol.).
[0483] Results
[0484] The results are presented in FIG. 6. Chosen cell surface
terminal N-acetyllactosamine (LN) units were studied as proof of
sialylation by N-glycan structural analyses (FIG. 6). Reaction
efficiency and level of terminal LN modification was followed by
MALDI-TOF mass spectrometric profiling of the neutral N-glycan
fraction. The chosen terminal LN units are marked with red arrows
in FIG. 6. The results clearly show that for the chosen terminal
LN, .alpha.2,3-sialylation is more efficient if the cells are
detached before the enzymatic reaction. The cell viability was not
affected by the modifications as compared to the control (0-)
cells.
Example 10
Optimal Modification Vessel and Applied Shear Force
[0485] The Isolation, Culture and Characterization of Human Cord
Blood-Derived MSCs (UCBMSC)
[0486] Cord blood was collected in a multiple bag system containing
17 ml of citrate phosphate dextrose buffer (Cord Blood Collection
System; Eltest, Bonn, Germany). Collections were performed at the
Helsinki University Central Hospital, Department of Obstetrics and
Gynaecology, and Helsinki Maternity Hospital. All donors gave
informed consent and the study protocol was approved by ethical
review board of Helsinki University Central Hospital and the
Finnish Red Cross Blood Service. Prior to the isolation of
mononuclear cells, the anti-coagulated cord blood was diluted 1:2
with 2 mM EDTA-PBS. Mononuclear cells were isolated using
Ficoll-Hypaque (Amersham Biosciences, Piscaway, N.J., USA) gradient
centrifugation. 1.times.10.sup.6/cm.sup.2 mononuclear cells were
plated on fibronectin (Sigma) coated tissue culture plates (Nunc)
in proliferation medium consisting of minimum essential medium
.alpha. (.alpha.MEM) with Glutamax (Gibco, Grand Island, N.Y., USA)
and 10% fetal calf serum (FCS) (Gibco) supplemented with 10 ng/mL
epidermal growth factor (EGF, Sigma), 10 ng/mL recombinant human
platelet-derived growth factor (rhPDGF-BB; R&D Systems,
Minneapolis, Minn., USA), 50 nM Dexamethasone (Sigma), 100 U/mL
penicillin+100 .mu.g/mL streptomycin (Invitrogen). The initial MSC
line establishment was performed in a humidified incubator with
hypoxic conditions (5% CO.sub.2, 3% O.sub.2 and 37.degree. C.).
[0487] Cells were allowed to adhere overnight and non-adherent
cells were washed out with medium changes. Proliferation media was
renewed twice a week. Established lines were passaged when almost
confluent and replated at 1000-3000 cells/cm.sup.2 in proliferation
media in normoxic conditions (5% CO.sub.2, 20% O.sub.2 and
37.degree. C.).
[0488] MSC Minimum Criteria Characterization
[0489] To ensure the MSC cell surface expression for critical MSC
markers, established MSC lines were analyzed for their cell surface
molecule expression by labeling with fluorochrome-conjugated
monoclonal antibodies: allophycocyanin (APC)-conjugated CD13 (BD
Pharmingen), phycoerythrin (PE)-conjugated CD14, CD19, CD34 and
CD45 (BD Pharmingen), fluorescein isothiocyanate (FITC)-conjucated
CD90 (clone 5E10, Stem Cell Technologies), FITC-CD105 (Abcam) and
FITC-HLA-DR (BD Pharmingen). Appropriate FITC-, PE-and
APC-conjugated isotypic controls (BD Biosciences) were used.
Labeling was carried out in 100 .mu.l of phosphate buffered saline
(PBS) with 0.5% ultra pure bovine serum albumin (BSA) on ice for 30
minutes. Flow cytometric analysis was performed on FACSAria (Becton
Dickinson Biosciences) with a 488-nm blue laser for (PE and FITC)
and a 633-nm red laser for (APC). Fluorescense was measured using
530/30-nm (FITC), 585/42-nm (PE) and 660/20-nm (APC) bandpass
filters. Data were analysed using FACSDiva software (BD
Biociences).Multipotent differentiation capacity was characterized
by inducing differentiation. 4-5.sup.th passage cells were cultured
in osteogenesis, chondrogenesis or adipogenesis inducing media up
to 3 weeks. Differentiation capacity was evaluated with standard
staining methods.
[0490] Enzymatic Cell Surface Glycomodification with Different
Incubation Options
[0491] The incubation options tested are presented in Table 4. The
incubation strategies were evaluated with and without an enzymatic
modification, in this case .alpha.2,3-sialyltransferase III (SAT)
modification (Calbiochem cat #566218, purification lot GF1061,
activity 5.6 mU/.mu.l) reaction for 2 hours together with CMP-NeuAc
(Kyowa Hakko, Tokyo) donor. UCBMSC 391P cells were used in these
studies in p4. .alpha.2,3-SATIII modifications were done with 100
mU enzyme and 1 mg CMP-NeuSAc donor in 300 .mu.l reaction buffer
(.alpha.MEM+0.5% human serum albumin HSA)/0.5.times.10e6 cells.
[0492] Analysis of the Glycomodified Cells after Different
Incubation Options
[0493] After the tested incubation options, cell viability was
determined by Trypan blue staining for each sample. The cells were
analyzed by flow cytometry for MSC minimum criteria cell
characterization markers as described above and for conjugated MAA
and MAL-1 plant lectin binding to determine the level of cell
surface .alpha.2,3-sialylation (a2,3SA). 1 .mu.l of the conjugated
.alpha.2,3SA-binding lectins MAA-FITC (Ey laboratories) and
MAL1-FITC (Vector laboratories, #FL-1311) was used per 1.times.10e5
cells with the same labeling protocol as described above for the
MSC minimum criteria panel. Flow cytometric analysis was performed
on FACSAria (Becton Dickinson Biosciences) with a 488-nm blue laser
for (PE and FITC) and a 633-nm red laser for (APC). Fluorescense
was measured using 530/30-nm (FITC), 585/42-nm (PE) and 660/20-nm
(APC) bandpass filters. Data were analysed using FACSDiva software
(BD Biociences).
[0494] Results and Discussion
[0495] Viability was not affected by any of the different
incubation strategies (Table 4). Adherence and aggregation of cells
can be reduced by using a conical tube instead of a cell culture
dish well (plastic is different, surface area is different).
Mechanical mixing by gentle pipetting every 30 min reduced
adherence significantly (FIG. 7). Enzymatic glycomodification was
most successful in a conical tube placed in a humidifed cell
incubator of tested options (Table 5).
[0496] Hypoxic (almost 0% O.sub.2) conditions, i.e. cells in tube
with closed cap, might quickly affect pH of the reaction buffer
used and thus affect enzyme activity. However, buffer-induced
alterations in cell surface sialylation levels are evident in
hypoxic conditions (Table 5).
[0497] The roughest cell incubation strategy, condition #5 i.e.
using a shaker at 150 rpm for 2 hours, seems to be too harsh since
changes were seen in the cell surface expression of the MSC minimal
criteria antigens (positive: CD13, CD44, CD49e, CD29, CD90, CD73,
CD105, HLA-ABC; negative: HLA-DR, CD14, CD19, CD34, CD45). Some
positive markers were less positive as compared to control cells
and some negative markers started to appear on the cell surface,
although viability with Trypan blue exclusion was >95% (Table
4). The results indicate that prolonged shaking might affect the
level of multipotency.
[0498] As a consensus, an open conical tube placed in a humidified
cell incubator with normoxic conditions and with mechanical mixing
every 30 min is optimal for enzymatic glycomodification
reactions.
Example 11
Human Serum Albumin (HSA) is an Optimal Supplement in Enzymatic
Glycomodifications
[0499] A. .alpha.2, 3-sialyltransferase assay
[0500] Materials and Methods
[0501] .alpha.2,3 SAT (2 mU; Calbiochem) was incubated with
lacto-N-neotetraose (LNnT) acceptor and 5 mM CMP-Neu5Ac at
37.degree. C. in minimum essential medium a (aMEM)(Gibco, Grand
Island, N.Y., USA) with 0.1% or without human serum albumin
(Albumin SPR, Sanquin, the Netherlands). Reaction times were 2
hours and overnight. Aliquots of the reaction mixtures were
subjected to size-exclusion chromatography on Superdex Peptide PC
3.2/30 column (GE Healthcare). The effluent was monitored with a UV
detector at 214 nm and the amount of the reaction product
Neu5Ac-LNnT was quantified by reference to monosaccharide standards
(GlcNAc and Neu5Ac).
[0502] Results and Discussion
[0503] Size-exclusion chromatography of the aliquots from
sialyltransferase assay indicated that .alpha.2,3 SAT was capable
of sialylating oligosaccharide acceptor LNnT in .alpha.MEM
containing human serum albumin. Analysis of aliquots taken from 2
hour reaction showed that 0.9 nmol of reaction product Neu5Ac-LNnT
was formed from 10 nmol of acceptor in .alpha.MEM containing
albumin while the enzyme was totally inactive in .alpha.MEM without
albumin. When the reaction was continued overnight, the product
peak could still be detected only from reaction where albumin was
present. (FIGS. 8A, 8B).
[0504] B. Desialylation of Human Stem Cells: Comparison between
Human or Bovine Albumin Supplements in Reaction Buffer
[0505] The Isolation, Culture and Characterization of Human Cord
Blood-Derived MSC (UCBMSC)
[0506] Cord blood was collected in a multiple bag system containing
17 ml of citrate phosphate dextrose buffer (Cord Blood Collection
System; Eltest, Bonn, Germany). Collections were performed at the
Helsinki University Central Hospital, Department of Obstetrics and
Gynaecology, and Helsinki Maternity Hospital. All donors gave
informed consent and the study protocol was approved by ethical
review board of Helsinki University Central Hospital and the
Finnish Red Cross Blood Service. Prior to the isolation of
mononuclear cells, the anti-coagulated cord blood was diluted 1:2
with 2 mM EDTA-PBS. Mononuclear cells were isolated using
Ficoll-Hypaque (Amersham Biosciences, Piscaway, N.J., USA) gradient
centrifugation. 1.times.10.sup.6/cm.sup.2 mononuclear cells were
plated on fibronectin (Sigma) coated tissue culture plates (Nunc)
in proliferation medium consisting of minimum essential medium
.alpha. (aMEM) with Glutamax (Gibco, Grand Island, N.Y., USA) and
10% fetal calf serum (FCS) (Gibco) supplemented with 10 ng/mL
epidermal growth factor (EGF, Sigma), 10 ng/mL recombinant human
platelet-derived growth factor (rhPDGF-BB; R&D Systems,
Minneapolis, Minn., USA), 50 nM Dexamethasone (Sigma), 100 U/mL
penicillin+100 .mu.g/mL streptomycin (Invitrogen). The initial MSC
line establishment was performed in a humidified incubator with
hypoxic conditions (5% CO.sub.2, 3% O.sub.2 and 37.degree. C.).
Cells were allowed to adhere overnight and non-adherent cells were
washed out with medium changes. Proliferation media was renewed
twice a week. Established lines were passaged when almost confluent
and replated at 1000-3000 cells/cm.sup.2 in proliferation media in
normoxic conditions (5% CO.sub.2, 20% O.sub.2 and 37.degree.
C.).
[0507] MSC Minimum Criteria Characterization
[0508] To ensure the MSC cell surface expression for critical MSC
markers, established MSC lines were analyzed for their cell surface
molecule expression by labeling with fluorochrome-conjugated
monoclonal antibodies: allophycocyanin (APC)-conjugated CD13 (BD
Pharmingen), phycoerythrin (PE)-conjugated CD14, CD19, CD34 and
CD45 (BD Pharmingen), fluorescein isothiocyanate (FITC)-conjucated
CD90 (clone 5E10, Stem Cell Technologies), FITC-CD105 (Abcam) and
FITC-HLA-DR (BD Pharmingen). Appropriate FITC-, PE-and
APC-conjugated isotypic controls (BD Biosciences) were used.
Labeling was carried out in 100 .mu.l of phosphate buffered saline
(PBS) with 0.5% ultra pure bovine serum albumin (BSA) on ice for 30
minutes. Flow cytometric analysis was performed on FACSAria (Becton
Dickinson Biosciences) with a 488-nm blue laser for (PE and FITC)
and a 633-nm red laser for (APC). Fluorescense was measured using
530/30-nm (FITC), 585/42-nm (PE) and 660/20-nm (APC) bandpass
filters. Data were analysed using FACSDiva software (BD
Biociences).Multipotent differentiation capacity was characterized
by inducing differentiation. 4-5.sup.th passage cells were cultured
in osteogenesis, chondrogenesis or adipogenesis inducing media up
to 3 weeks. Differentiation capacity was evaluated with standard
staining methods.
[0509] Desialylation Reactions
[0510] Human UCBMSCs were desialylated for 2 hours in conical tubes
in a 37.degree. C. cell incubator with 200 mU Vibrio cholerae
neuraminidase per 1.times.10e6 cells in 600 .mu.l .alpha.MEM+0.5%
human serum albumin (HSA) (Albumin SPR, Sanquin) or ultrapure
(>99%) bovine serum albumin (BSA) (Sigma). The cells were mixed
by mechanical suspension every 30 min.
[0511] Validation of Glycomodification by Flow Cytometry
[0512] MSC minimum criteria characterization was performed as
described above. Cell surface lectin binding profile was studied
with FITC- or biotin conjugated MAA, MAL-1 or SNA plant lectins
before and after enzymatic glycomodifications. FITC-conjugated
streptavidin secondary antibody staining was done when using
biotin-conjugated lectins. The following Lewis X (Lex)/sialyl Lewis
X (sLex) glycoform specific antibodies were also used: CD15 (TG-1)
for Lex (BD Pharmingen), PSGL-1 (core 2 0-glycan) for sLex (R&D
Systems) and CD15s for sLex (BD Pharmingen). 1.times.10.sup.5 cells
were labeled in Ca.sup.2+-free PBS supplemented with 0.5-1% BSA.
Flow cytometric analysis was performed on FACSAria (Becton
Dickinson Biosciences) with a 488-nm blue laser for (PE and FITC)
and a 633-nm red laser for (APC). Analysis was performed using the
FACSDiva software (Beckton Dickinson)
[0513] Results and Discussion
[0514] The results are presented in Table 6. Control1 indicates
cells at time point 0, analyzed directly after trypsinization.
Control2 indicates reaction buffer incubated cells with no enzyme.
Desialylation for 2 hours with V.cholera sialidase efficiently
reduces binding of both the .alpha.2,3-sialic acid binding lectins
MAA and MAL1 and .alpha.2,6-sialic acid binding lectin SNA with an
over 20% more effective result in reactions supplemented with HSA
than BSA (Table 6). However, the binding of the sLex-binding
glycoform antibody CHO-131 is reduced more when the reaction has
been supplemented with BSA as compared with HSA. Interestingly, the
BSA supplemented reaction induces more spontaneous changes in cell
surface .alpha.2,3-sialylation levels as studied by MAA and MAL
lectin binding (Table 6, control1 versus control2). This also
indicates that HSA stands out as a better supplement than BSA,
since the levels of "spontaneous" cell surface sialylation induced
by buffer only are more moderate in HSA supplemented reactions. In
conclusion, the results clearly indicate than HSA is a superior
choice as a supplement in glycomodification reactions of human stem
cell. Supplementing the glycomodification buffer with HSA also
gives a xeno-free glycomodification buffer for stem cells.
Example 12
MSC Optimal Suspension Density
[0515] Materials and Methods
[0516] Human umbilical cord blood-derived mesenchymal stem cells
(UCBMSC) were detached with TrypLE (Invitrogen) and incubated as
suspension cells in .alpha.MEM+0.5% human serum albumin (HSA)
(Albumin SPR, Sanquin, the Netherlands) either with 0.5.times.10e6
(1.7.times.10e6 cells/ml) or 1.times.10e6 cells/300 .mu.l
(3.3.times.10e6 cells/ml) cell densities. The cells were kept in
conical tubes with open caps in a humidified cell incubator for 2
hours with a mechanical mixing by pipetting every 30 min. At the
end of the incubation the cells were transferred to cell culture
dishes and were photographed by phase contrast microscopy.
[0517] Results and Discussion
[0518] FIG. 9 demonstrates clearly that a suspension cell density
of 1.times.10e6 UCBMSCs in 300 .mu.l (3.3.times.10e6/ml) is too
high for UCBMScs since a lot of small cell aggregates are formed
although repeated mechanical mixing. An optimal cell density seems
to be 0.5.times.10e6 UCBMSCs cells in 300 .mu.l (1.7.times.10e6/ml)
as demonstrated in FIG. 10. It is noteworthy that bone
marrow-derived MSCs (BMIVISCs) can tolerate more dense suspension
incubations than UCBMSCs.
Example 13
Mild Cell Dissociation Preceding Enforced Enzymatic
Glycomodifications
[0519] Materials and Methods
[0520] Human bone marrow-derived mesenchymal stem cells (BMMSC)
(168 p'7) and human umbilical cord blood-derived mesenchymal stem
cells (UCBMSC) (391P p4) were detached from culture plates with
either porcine Trypsin-0.25% EDTA (Gibco Invitrogen) or TrypLE
Express (Gibco Invitrogen, #12605-010) according to the
manufacturer's instructions. The cell detachment process was
observed by microscopy and stopped with complete culture media when
complete. Detached cells were analyzed by flow cytometry by
staining 1.times.10e5 cells in PBS (pH 7.2) and 0.3% BSA with 1
.mu.l of the conjugated plant lectins MAA-FITC, MAL1-biotin
(+streptavidin-FITC secondary antibody staining) and SNA-FITC for
30 min on ice. Flow cytometric analysis was performed on FACSAria
(Becton Dickinson Biosciences) with a 488-nm blue laser for (PE and
FITC) and a 633-nm red laser for (APC). Fluorescense was measured
using 530/30-nm (FITC), 585/42-nm (PE) and 660/20-nm (APC) bandpass
filters. Data were analysed using FACSDiva software (BD
Biociences).
[0521] Results and Discussion
[0522] Standard porcine Trypsin-0.25% EDTA dissociation of MSCs was
compared to TrypLE Express (Invitrogen) dissociation. Tryple
Express is a recombinant enzyme derived from microbial
fermentation, formulated in D-PBS with 1 mM EDTA and offers an
improved stability, lower cost and animal-free, protease-free
origin compared to standard porcine trypsin. According to the
manufacturer, the use of porcine trypsin or TrypLE is similar and
cell detachment equal. In our experiments, TrypLE detachment could
be performed in 4 minutes, working similar to porcine trypsin, and
the cell viability after detachment was very high with both
reagents. The lectin labeling of UCBMSC was similar after porcine
Trypsin vs. Tryple detachment (FIGS. 10A, 10B). BMMSC had higher
MAL-1 and SNA binding after TrypLe detachment, suggesting possibly
better preservation of cell surface sialic acid glycostructures on
cell surface proteins. In conclusion, TrypLE preserves the cell
surface sialylation levels at least as well as standard porcine
trypsin and due to the other benefits of TrypLE Express, TrypLE
Express stands out as an excellent choice for mild MSC cell
dissociation preceding subsequent in vitro glycomodifications.
Example 14
Flow Cytometric Analysis Platform for Cell Surface Sialyl- and
Sialyl-Fucosylmodifications
[0523] A flow cytometry-based platform for fast and minimum sample
consuming validation of the success of enforced in vitro
glycomodifications was developed, especially for changes in cell
surface levels of .alpha.2,3-sialylation and
.alpha.1,3-fucosylation. In the absence of validated antibodies
against sialic acids, the flow cytometric analyses were based on
labeling with conjugated plant lectins (Table 7). The specificity
of MAA-lectins, which should only recognize .alpha.2,3-linked
sialic acid, varies remarkably between different manufacturers and
conjugations, thus binding of several MAA lectins were compared and
studied thoroughly with serial dilutions. Similarly, other sialic
acid recognizing lectins SNA and LFA and terminal galactose
recognizing ECA were examined. The secondary label
streptavidin-FITC (eBiosciences, #11-4317) was used with
biotin-conjugated lectins. .alpha.1,3-fucosylated glycan structures
are validated with the following antibodies (also presented in
Table 8): PSGL-1, clone CHO131 (GF526/VPUO37) (R&D systems),
CD15/TG-1 (GF525) (abcam), CD15s/CSLEX1 (VPUO20) (BD Pharmingen),
HECA-452 (CLA) (BD Pharmingen #555947). The cells were fixed in
0.5% paraformaldehyde (PFA) before stainings with plant lectins to
avoid biological effects of the lectins. All stainings were
performed with PBS (pH 7.2)+0.5% BSA for 30 minutes on ice and with
appropriate subsequent secondary antibody stainings as needed.
Example 15
Efficient Desialylation Protocol of Stem Cells
[0524] The isolation, culture and characterization of human cord
blood-derived MSC (UCBMSC) Cord blood was collected in a multiple
bag system containing 17 ml of citrate phosphate dextrose buffer
(Cord Blood Collection System; Eltest, Bonn, Germany). Collections
were performed at the Helsinki University Central Hospital,
Department of Obstetrics and Gynaecology, and Helsinki Maternity
Hospital. All donors gave informed consent and the study protocol
was approved by ethical review board of Helsinki University Central
Hospital and the Finnish Red Cross Blood Service. Prior to the
isolation of mononuclear cells, the anti-coagulated cord blood was
diluted 1:2 with 2 mM EDTA-PBS. Mononuclear cells were isolated
using Ficoll-Hypaque (Amersham Biosciences, Piscaway, N.J., USA)
gradient centrifugation. 1.times.10.sup.6/cm.sup.2 mononuclear
cells were plated on fibronectin (Sigma) coated tissue culture
plates (Nunc) in proliferation medium consisting of minimum
essential medium .alpha. (.alpha.MEM) with Glutamax (Gibco, Grand
Island, N.Y., USA) and 10% fetal calf serum (FCS) (Gibco)
supplemented with 10 ng/mL epidermal growth factor (EGF, Sigma), 10
ng/mL recombinant human platelet-derived growth factor (rhPDGF-BB;
R&D Systems, Minneapolis, Minn., USA), 50 nM Dexamethasone
(Sigma), 100 U/mL penicillin+100 .mu.g/mL streptomycin
(Invitrogen). The initial MSC line establishment was performed in a
humidified incubator with hypoxic conditions (5% CO.sub.2, 3%
O.sub.2 and 37.degree. C.). Cells were allowed to adhere overnight
and non-adherent cells were washed out with medium changes.
Proliferation media was renewed twice a week. Established lines
were passaged when almost confluent and replated at 1000-3000
cells/cm.sup.2 in proliferation media in normoxic conditions (5%
CO.sub.2, 20% O.sub.2 and 37.degree. C.).
[0525] MSC Minimum Criteria Characterization
[0526] To ensure the MSC cell surface expression for critical MSC
markers, established MSC lines were analyzed for their cell surface
molecule expression by labeling with fluorochrome-conjugated
monoclonal antibodies: allophycocyanin (APC)-conjugated CD13 (BD
Pharmingen), phycoerythrin (PE)-conjugated CD14, CD19, CD34 and
CD45 (BD Pharmingen), fluorescein isothiocyanate (FITC)-conjucated
CD90 (clone 5E10, Stem Cell Technologies), FITC-CD105 (Abcam) and
FITC-HLA-DR (BD Pharmingen). Appropriate FITC-, PE-and
APC-conjugated isotypic controls (BD Biosciences) were used.
Labeling was carried out in 1000 of phosphate buffered saline (PBS)
with 0.5% ultra pure bovine serum albumin (BSA) on ice for 30
minutes. Flow cytometric analysis was performed on FACSAria (Becton
Dickinson Biosciences) with a 488-nm blue laser for (PE and FITC)
and a 633-nm red laser for (APC). Fluorescense was measured using
530/30-nm (FITC), 585/42-nm (PE) and 660/20-nm (APC) bandpass
filters. Data were analysed using FACSDiva software (BD
Biociences).Multipotent differentiation capacity was characterized
by inducing differentiation. 4-5.sup.th passage cells were cultured
in osteogenesis, chondrogenesis or adipogenesis inducing media up
to 3 weeks. Differentiation capacity was evaluated with standard
staining methods.
[0527] Desialylation and Validation of Glycomodification
[0528] UCBMSCs (391P p6) were desialylated with 200 mU Vibrio
cholerae sialidase/0.5.times.10e6 in 300 .mu.l of .alpha.MEM
Glutamax+0.5% human serum albumin (HSA) or 0.5% bovine serum
albumin (BSA) for 2 h at 37.degree. C. with mixing by pipetting
every 30 min. Desialylation was stopped by adding excess reaction
buffer and centrifugation. Cell surface lectin binding profile of
the UCBMSCs was studied with FITC- or biotin conjugated MAA, MAL-1
or SNA lectins before and after enzymatic glycomodifications.
FITC-conjugated streptavidin secondary antibody staining was done
when using biotin-conjugated lectins. Sialic acid-dependent binding
of the sLex glycoform antibody CHO-131 (PSGL-1 core 2 o-glycan) was
also used together with appropriate conjugated secondary antibody.
1.times.10.sup.5 BM-MSC:s were labeled per in Ca.sup.2+-free PBS
supplemented with 1% BSA. Analysis was performed using the FACSDiva
software (Beckton Dickinson)
[0529] Results and Discussion
[0530] Desialylation of CB MSCs (391P p6) for 2 h in .alpha.MEM
Glutamax+0.5% HSA showed significant reduction of MAA, MALI, SNA
and CHO131 (VPUO37) binding (FIGS. 11A-11D). Desialylation was more
efficient in .alpha.MEM+0.5% HSA than .alpha.MEM+0.5% BSA (FIGS.
11A-11D). CHO131 (VPUO37) staining was lower in cells desialylated
in .alpha.MEM Glutamax+0.5% BSA. The difference in desialylation
between incubation media correlates with reaction buffer-induced
MAA and MAL1 staining that is significantly higher in .alpha.MEM
supplemented with 0.5% BSA. Desialylation using Vibrio cholerae
sialidase was significantly more efficient than desialylation with
Clostridium perfringens sialidase.
Example 16
Cell Surface Enzymatic Modification of Mesenchymal Stem Cells in
Different Cell Culture Media
[0531] Materials and Methods
[0532] The Isolation, Culture and Characterization of Human Cord
Blood-Derived MSC (UCBMSC)
[0533] Cord blood was collected in a multiple bag system containing
17 ml of citrate phosphate dextrose buffer (Cord Blood Collection
System; Eltest, Bonn, Germany). Collections were performed at the
Helsinki University Central Hospital, Department of Obstetrics and
Gynaecology, and Helsinki Maternity Hospital. All donors gave
informed consent and the study protocol was approved by ethical
review board of Helsinki University Central Hospital and the
Finnish Red Cross Blood Service. Prior to the isolation of
mononuclear cells, the anti-coagulated cord blood was diluted 1:2
with 2 mM EDTA-PBS. Mononuclear cells were isolated using
Ficoll-Hypaque (Amersham Biosciences, Piscaway, N.J., USA) gradient
centrifugation. 1.times.10.sup.6/cm.sup.2 mononuclear cells were
plated on fibronectin (Sigma) coated tissue culture plates (Nunc)
in proliferation medium consisting of minimum essential medium a
(aMEM) with Glutamax (Gibco, Grand Island, N.Y., USA) and 10% fetal
calf serum (FCS) (Gibco) supplemented with 10 ng/mL epidermal
growth factor (EGF, Sigma), 10 ng/mL recombinant human
platelet-derived growth factor (rhPDGF-BB; R&D Systems,
Minneapolis, Minn., USA), 50 nM Dexamethasone (Sigma), 100 U/mL
penicillin+100 .mu.g/mL streptomycin (Invitrogen). The initial MSC
line establishment was performed in a humidified incubator with
hypoxic conditions (5% CO.sub.2, 3% 0.sub.2 and 37.degree. C.).
Cells were allowed to adhere overnight and non-adherent cells were
washed out with medium changes. Proliferation media was renewed
twice a week. Established lines were passaged when almost confluent
and replated at 1000-3000 cells/cm.sup.2 in proliferation media in
normoxic conditions (5% CO.sub.2, 20% O.sub.2 and 37.degree.
C.).
[0534] MSC Minimum Criteria Characterization
[0535] To ensure the MSC cell surface expression for critical MSC
markers, established MSC lines were analyzed for their cell surface
molecule expression by labeling with fluorochrome-conjugated
monoclonal antibodies: allophycocyanin (APC)-conjugated CD13 (BD
Pharmingen), phycoerythrin (PE)-conjugated CD14, CD19, CD34 and
CD45 (BD Pharmingen), fluorescein isothiocyanate (FITC)-conjucated
CD90 (clone 5E10, Stem Cell Technologies), FITC-CD105 (Abcam) and
FITC-HLA-DR (BD Pharmingen). Appropriate FITC-, PE-and
APC-conjugated isotypic controls (BD Biosciences) were used.
Labeling was carried out in 100 .mu.l of phosphate buffered saline
(PBS) with 0.5% ultra pure bovine serum albumin (BSA) on ice for 30
minutes. Flow cytometric analysis was performed on FACSAria (Becton
Dickinson Biosciences) with a 488-nm blue laser for (PE and FITC)
and a 633-nm red laser for (APC). Fluorescense was measured using
530/30-nm (FITC), 585/42-nm (PE) and 660/20-nm (APC) bandpass
filters. Data were analysed using FACSDiva software (BD
Biociences).Multipotent differentiation capacity was characterized
by inducing differentiation. 4-5.sup.th passage cells were cultured
in osteogenesis, chondrogenesis or adipogenesis inducing media up
to 3 weeks. Differentiation capacity was evaluated with standard
staining methods.
[0536] .alpha.2,3-sialylation in different cell culture media
[0537] The following reaction buffers were tested: a) minimum
essential medium .alpha. (aMEM) (Gibco, Grand Island, N.Y.,
USA)+0.5% human serum albumin (HSA) (Albumin SPR, Sanquin, the
Netherlands), b) xeno-free StemPro MSC SFM (Invitrogen)
supplemented with 0.1% StemPro SFM XF (Invitrogen) and c)
Ca.sup.2+/Mg.sup.2+-free Hank's balanced salt solution (HBSS)+2 mM
Hepes+0.1% HSA (Sackstein et al. Nature Genetics 2008). The
incubation strategies were evaluated with and without an enzymatic
modification, in this case .alpha.2,3-sialyltransferase III (SAT)
modification (Calbiochem cat#566218) reaction for 2 hours together
with CMP-NeuAc (Kyowa Hakko, Tokyo) donor. .alpha.2,3-SATIII
modifications were done with 100 mU enzyme and 1 mg CMP-Neu5Ac
donor in 300 .mu.l reaction buffer/0.5.times.10e6 cells. The
incubation time was set to 2 h or 40 min for the HBSS buffer
version (Sackstein et al. Nature Genetics 2008). Incubations were
performed in 15 ml conical tubes (cap open) in +37.degree. C.
incubator with mechanical mixing by pipetting every 30 minutes.
[0538] Analysis of the Glycomodified Cells
[0539] After the tested incubation options, cell viability was
determined by Trypan blue staining for each sample. The cells were
analyzed by flow cytometry for MSC minimum criteria cell
characterization markers as described above and for conjugated MAA,
MAL-1 and SNA plant lectin binding to determine the levels of cell
surface .alpha.2,3 and .alpha.2,6-sialylation. 1 .mu.l of the
conjugated lectins were used per 1.times.10e5 cells with the same
labeling protocol as described above for the MSC minimum criteria
panel. Flow cytometric analysis was performed on FACSAria (Becton
Dickinson Biosciences) with a 488-nm blue laser for (PE and FITC)
and a 633-nm red laser for (APC). Fluorescense was measured using
530/30-nm (FITC), 585/42-nm (PE) and 660/20-nm (APC) bandpass
filters. Data were analysed using FACSDiva software (BD
Biociences).
[0540] Results and Discussion
[0541] After 2 h incubation, >95% viability (Trypan blue
staining) was detected in each incubation buffer. An increase in
cell surface .alpha.2,3-sialylation levels after enforced
.alpha.2,3-sialylation with .alpha.2,3-sialyltransferase III (SAT)
was highest in .alpha.MEM+0.5% HSA reaction buffer (Table 9).
However, an increase in cell surface .alpha.2,3-sialylation was
also evident in StemPro MSC XF buffer (Table 9). Incubation related
increase of MAA-FITC and MALI-FITC lectin binding was highest in
StemPro MSC XF+0.1% StemPro SFM XF supplements and very low in 40
min incubation in HBSS buffer.
Example 17
Targeting of Stem Cells to Bone Marrow by Glycomodification
[0542] Materials and Methods
[0543] Cells, Labeling, Glycomodification and Validation
[0544] Bone Marrow (BM) Derived Mesenchymal Stem Cells (MSC)
[0545] BMIVISC:s were obtained as described by Leskela et al.
(2003). Briefly, bone marrow-derived mononuclear cells were
cultured in Minimum Essential Alpha-Medium (.alpha.MEM)
supplemented with 20 mM HEPES, 10% fetal calf serum,
penicillin-streptomycin and 2 mM L-glutamine (all from Gibco).
After a cell attachment period of 2 days the cells were washed with
PBS, subcultured further by plating the cells at a density of
2000-3000 cells/cm.sup.2 in the same media and replacing the medium
twice a week until near confluence. The cells used in the example
were between passage 2-5.
[0546] Cell Labeling for in vivo Tracking
[0547] BMMSCs were detached with 0.25% trypsin/1 mM EDTA in
Ca.sup.2+/Mg.sup.2+-free PBS (Invitrogen) for 3 minutes. The
trypsinization was inhibited by adding excess of complete culture
media. Cell viability and cell amounts were determined with trypan
blue exclusion. The cells were metabolically labelled with 10 MBq
.sup.3H-2-deoxy-D-glucose ([.sup.3H]-2DG) per 1.times.10.sup.6
cells or labelled with Tc99-HMPAO. Labeling efficiency was
determined by measuring radioactivity of the supernatants in every
step of the labeling protocols and aliquots of the labelled cells
or the final cell pellets. The .sup.3H-2-deoxy-D-glucose
([.sup.3H]-2DG) metabolic labeling protocol yielded a specifc
activity of 6.times.10.sup.6 cpm/1.times.10.sup.6 cells.
[0548] Enforced .alpha.1,3-fucosylation of BMMSCs
[0549] The labeled cells were recounted after the labeling
procedure and cell-specific standard curves with specific cell
amounts were prepared for subsequent radioactivity measurements.
The rest of the cells were centrifuged 300.times.g for 5 min, the
supernatant was completely removed and 1.times.106 cells were
resuspended in 300 .mu.l enzyme reaction buffer composed of
.alpha.MEM supplemented with 0.5% human serum albumin (HSA)
(Albumin SPR, Sanquin, the Netherlands). 1.times.10e6 cells were
enzymatically cell surface .alpha.1,3-fucosylated with 15 mU human
recombinant (Spodoptera frugiperda) .alpha.1,3-Fucosyltransferase
VI (FUTVI) (Calbiochem) and 1 mg GDP-fucose in 600 .mu.l reaction
buffer for 1-2 hours at +37.degree. C. The original FUTVI enzyme
storage buffer was exchanged to the reaction buffer and activity
examined before the experiment. To prevent cell aggregation or cell
attachment to the modification vessel, the reactions were mixed by
mechanical pipetting every 30 minutes during the incubation.
Parallel reactions with cells only in the reaction buffer without
enzymes were always included in each experiment and used as control
cells in the subsequent in vivo experiments. The enforced enzymatic
.alpha.1,3-fucosylation was stopped by adding excess volume of
reaction buffer and washing the cells twice with reaction buffer.
Aliquotes were taken from the cell reactions to monitor cell
viability by Trypan blue exclusion and for validation of the
success of the applied enzymatic glycomodification by flow
cytometry.
[0550] Validation of Glycomodifications
[0551] The anti-Lex/sLex glycoform antibodies used for FACS
analysis were: CHO-131 (PSGL-1 sLex on core II O-glycans, R&D
Systems), CSLEX (CD15s, BD Pharmingen), FITC-HECA 452 (CLA, BD
Pharmingen) and TG-1 (CD15, abcam). 1.times.10.sup.5 0.5% PFA fixed
BMMSCs were labelled with 3 .mu.l of unconjugated CHO-131, CSLEX
and TG-1 antibodies and 2 .mu.l of FITC-conjugated HECA 452
antibody in Ca.sup.2+-free PBS supplemented with 0.5% BSA for 30
minutes on ice and protected from light. Alexa-Fluor 488 conjugated
goat anti-mouse IgG (Molecular Probes) was used in 1:500 dilution
for secondary antibody stainings for the unconjugated primary
antibodies for 20 min on ice protected from light. The samples were
washed in excess PBS+0.5% BSA and analysed with FACSAria (Beckton
Dickson) flow cytometer. Analysis was performed using the FACSDiva
software (Beckton Dickinson).
[0552] Animals and in vivo Biodistribution Detection of Labelled
Stem Cells
[0553] Adult (7-10 weeks) male (with [.sup.3H]-2DG labelled cells)
or female (with Tc99-HMPAO labelled cells), age-matched Hsd:Athymic
Foxn1nu (Harlan) mice were used in the in vivo experiments. The
acclimatization period was always at least 7 days before the
experiments. The animal room temperature was 21.+-.2.degree. C. and
humidity was between 40-60%. Lightning was artificial, 12 h light
and 12 h dark. The mice were provided with irradiated fodder and
normal tap water ad libitum. No formal randomization or grouping
was done. Animals were randomly allocated to the study groups. The
cells were centrifuged and resuspended in 0.9% NaCl in a cell
concentration of 5.times.10e6 cells/ml. 0.5.times.10e6 cells
(Tc99-labelled) or 1.times.10e6 cells ([.sup.3H]-2DG labelled) were
injected intravenously (i.v) in the tail vein in 100 .mu.l 0.9%
NaCl. After 1 and 12 hours, the animals were sacrificed and tissues
samples were prepared. Bone marrow was collected from femoral bones
in 1 ml 0.9% NaCl. Radioactivity was measured by .sup.3H or gamma
counting (Wallac, Finland).
[0554] Results and Discussion
[0555] The staining of BMMSCs with and without enzymatic in vitro
fucosylation with antibodies against sialyl Lewis x (sLex) and
Lewis x (Lex) epitopes is presented in Table 10. The anti-sLex
antibodies CSLEX and HECA-452 and the anti-Lex antibody TG-1 stain
the native cells only minimally, whereas CHO-131 stains 52% of the
native cells. Labeling with all of the three antibodies increases
when the cells are enzymatically .alpha.1,3-fucosylated indicating
a very successful cell surface .alpha.1,3-fucosylation. The results
also indicate, that although all three antibodies recognize
fucosylated epitopes, CHO-131 binding is less dependent on fucose
than the binding of the other anti-sLex and anti-Lex
antibodies.
Example 18
Effect of Enzymatic Cell Surface Glycomodification on MSC
Proliferation Behavior
[0556] Materials and Methods
[0557] Umbilical cord blood-derived mesenchymal stem cells (UCBMSC)
in 70% confluency were detached with TrypLE Express (Gibco) for 6
minutes. The cell detachment was stopped by adding excess of
.alpha.MEM supplemented with 10% human serum albumin (HSA) (Albumin
SPR, Sanquin, the Netherlands). Viability of the detached UCBMSCs
was studied by Trypan blue exclusion. The detached cells were
centrifuged 300.times.g for 4 min, the supernatant was completely
removed and cells were resuspended in enzyme reaction buffer
composed of Minimum Essential Medium (MEM) a medium supplemented
with 0.5% HSA (reaction buffer control).
[0558] 1.times.10.sup.6 cells per reaction were incubated with
glycosylation enzymes and sugar donors in the following way: 200 mU
Neuraminidase from Vibrio cholerae for 1 h, 200 mU rat recombinant
(Spodoptera frugiperda) .alpha.2,3-(N)-Sialyltransferase III (SAT)
(Calbiochem) and 1 mg CMP-Neu5Ac for 1 h, Neuraminidase from Vibrio
cholerae for 1 h followed by SAT and 1 mg CMP-Neu5Ac for the same
cells for 1 h. The neuraminidase treated cells that were also
treated with SAT, were washed twice with PBS between the
treatments. Parallel reactions with cells only in the reaction
buffer without the enzymes were also included in the experiment.
Prior to the experiment, the original enzyme buffers were exchanged
to the above mentioned reaction buffer with Microcon Ultracel YM10
(Millipore). The UCB-MSC cell suspensions were incubated in
+37.degree. C., 5% CO.sub.2 cell incubator in conical tubes (15 ml
Falcon tubes, Nunc). To prevent cells to re-adhere to each others
or surface of the tubes, the reactions were mixed by mechanical
pipetting using ART 1000E filter tips (Molecular BioProducts) with
M1000 micropipette (Biohit) every 30 minutes during the incubation.
When the reactions were collected the number of cells and cell
viability were determined.
[0559] After the treatments cells from each reaction were plated
1000 cells per cm.sup.2 and incubated in +37.degree. C., 5%
CO.sub.2 cell incubator in proliferation medium (Minimum Essential
Medium (MEM) .alpha. medium supplemented with 10% fetal calf serum
(Gibco), 50 nM dexamethasone (Sigma), 10 ng/ml EGF (Sigma), 10
ng/ml rhPDGF-BB (R&D), 100 U/ml penicillin (Gibco), 1%
streptomycin (Gibco)). As a control, proliferating cells without
any treatments were plated 1000 cells per cm.sup.2. After six days
nearly confluent cells were detached with TrypLE Express (Gibco)
and the number of cells was determined.
[0560] Results and Discussion
[0561] Cell viability was unchanged after the enzymatic
glycomodification and always >90%. No changes were seen in the
proliferation behaviour and morphology of in vitro cell surface
glycomodified MSCs, when the reaction buffer consists of cell
culture media supplemented with 0.5% HSA (Table 11 and FIG.
13).
Example 19
Analysis of Suitable Medium
[0562] Materials and Methods
[0563] Materials: StemPro MSC SFM and StemPro MSC SFM+XF,
100.times. Supplement (Invitrogen; Prante et al., 2009, "A Closed,
Xenogeneic-Free Isolation and Expansion System for Human
Mesenchymal Stromal Cells", Abstract at the 3rd International
workshop on multipotent stromal cells (MSCs) for regenerative
medicine and immune regulation, Frankfurt, Germany.)
[0564] Glycan isolation: 10 .mu.l of 100.times.XF Supplement is
subjected to N-glycosidase F digestion in 1000 total reaction
volume (Nyman et al. 1998 Eur. J. Biochem.). Similarly, potential
glycoproteins are analyzed from precipitation by ice-cold acetone
(Verostek et al.) of 37.5 .mu.l of medium XF supplemented xenofree
medium in 50 .mu.l of total reaction volume. Sialylated N-glycans
are isolated by graphitized carbon microcolumn solid-phase
extraction (Hemmoranta et al. 2007 Exp. Hematol.).
[0565] Glycan analysis: Sialylated N-glycans are analyzed by
MALDI-TOF MS in negative ion linear mode (Heiskanen et al.
Glycoconj. J. in press). Neu5Gc content is analyzed by calculating
relative intensities of indicator signals (m/z 1946, 2237, and
2256) and Neu5Ac indicator signals (m/z 1930 and 2221/2222)
(Heiskanen et al. 2007 Stem Cells). Also neutral N-glycans are
analyzed by MALDI-TOF MS in positive ion reflector mode.
[0566] N-Glycan Analysis of a Cell Culture Medium Suitable for
Glycomodification and its Components
[0567] We have earlier reported on Neu5Gc in different cell culture
media. Invitrogen has developed a new cell culture medium which is
claimed to be serum free and free from any kind of animal derived
material.
[0568] Sialylated N-glycans are analyzed by MALDI-TOF MS in
negative ion linear mode. Neu5Gc content was analyzed by
calculating relative intensities of indicator signals (m/z 1946,
2237, and 2256) and Neu5Ac indicator signals (m/z 1930 and
2221/2222). Neu5Gc indicator signals were not found either from
medium or supplement.
[0569] Signals from acidic N-glycans of StemPro MSC SFM XF,
100.times. Supplement are observed. Main peaks are S2H5N4 and
S1H5N4 (FIG. 14). S1H5N4 and S2H5N4 are typical human transferrin
N-glycan monosaccharide compositions. Regarding the other N-glycan
structures that were found, human serum transferrin has been
reported to contain trace amounts of fucosylation and the relative
proportions of bi- and triantennary structures are 9:1 (Spik et al.
1988, Biochimie 70:1459-69).
[0570] Also neutral N-glycans are analyzed. However, no neutral
N-glycans are detected. Three acidic N-glycans (S1H5N4, S1H5N4F1
and S2H5N4) partially leak into neutral fraction because of huge
amount of these glycans compared to neutral glycans. In StemPro MSC
SFM neither neutral nor acidic N-glycans are found.
TABLE-US-00003 TABLE 1 Table 1. Used antibodies and lectins in FACS
analysis of control and glycomodified BM-MSC:s. MAA (Maackia
amurensis) binds to a2,3-linked sialic acids
(Neu5Ac/Gc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-R), SNA (Sambucus
nigra) to .alpha.2,6-linked sialic acids (Neu5Ac.alpha.2-
6Gal(NAc)-R). RCA (Ricinus communis) and ECA (Erythrina
cristagalli) binds mainly to Gal.beta.1-4GlcNAc.beta.1-R. MSC
immunophenotype antibodies Plant lectins Glycoform antibodies
Positive markers: MAA-FITC Lex GF517 CD90, CD105, CD73 MAA-biotin
(2.degree.ab (core 2 O-glycan) sLex HLA-ABC streptavidin FITC)
GF526 Negative markers: SNA-FITC (sLex/CSLEX1) GF516, CD14, CD19,
CD34, SNA-biotin (2.degree.ab VPU020 CD45 HLA-DR streptavidin FITC)
(FITC or PE conjugated) RCA-FITC ECA-biotin (2.degree.ab
streptavidin FITC)
TABLE-US-00004 TABLE 2 Viability of BM-MSC after trypsinization in
different conditions Number of BM-MSCs in 300 .mu.l cell
suspension. After 2 hours incubation in indicated conditions, the
cells still in suspension were collected and their number
calculated. The cells adhered to cell culture vessel bottom were
not collected. Viability was determined by Trypan blue exclusion.
There were less cells left in suspension after 2 h incubation in
buffer containing divalent cations (.alpha.-MEM + 0.5% HSA), due to
adherence to cell culture vessel if the reaction was not
continuously resuspended. after 2 h after 2 h before incubation no
resuspension with resuspension .alpha.MEM + 0.5% HSA live cells 175
000 87 000 105 000 dead cells 0 0 0 viability 100% 100% 100% HBSS
(Ca.sup.2+/Mg.sup.2+-free) + 0.1% HSA live cells 175 000 94 500 91
500 dead cells 0 3000 1500 viability 100% 97% 98%
TABLE-US-00005 TABLE 3 Sulfo-specific antibodies Specificity
Antibody (clone) name Comments Group 1. Binds sulfo, no Sialic
acid, no Fuc 1a. Specific antibodies M-DC8
Gal(.beta.1-4)[HSO3(-6)]GlcNAc(.beta.1-3)Gal(.beta.1-4)Glc(.beta.1-)-
-R [1], [3] DD2
Gal(.beta.1-4)[HSO3(-6)]GlcNAc(.beta.1-3)Gal(.beta.1-4)Glc(.beta.1-)-R
[1], [3] 1b. Other antibodies DD1*
Gal(.beta.1-4)[HSO3(-6)]GlcNAc(.beta.1-3)Gal(.beta.1-4)Glc(.beta.1-)--
R [1], [3]
[HSO3(-6)]Gal(.beta.1-4)[HSO3(-6)]GlcNAc(.beta.1-3)Gal(.beta.1-4)Glc(.bet-
a.1-)-R
[HSO3(-6)]Gal(.beta.1-4)[HSO3(-6)]GlcNAc(.beta.1-3)[HSO3(-6)]Gal(.beta.1--
)-R *Miltenyi data sheet for Ab M-DC8 indicates this as clone name
Group 2. Binds sulfo and sialic acid, but no Fuc 2a. Specific
antibodies KN343
Neu5Ac(.alpha.2-6)Gal(.beta.1-4)[HSO3(-6)]GlcNAc(.beta.1-)-R [8]
Group 3. Binds sulfo and Fuc, but no sialic acid 3a. Specific
antibodies AG273
Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-6)]GlcNAc(.beta.1-3)Gal(.beta.-
1-)-R [1] AG97
Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-6)]GlcNAc(.beta.1-3)Gal(.beta.1-
-)-R [1] F2 HSO3(-3)Gal(.beta.1-3)[Fuc(.alpha.1-4)]GlcNAc-R [1],
[4] 91.9H
HSO3(-3)Gal(.beta.1-3)[Fuc(.alpha.1-4)]GlcNAc(.beta.1-3)Gal-R [1]
3b. Other antibodies AG107
Gal(.beta.1-4)[HSO3(-6)]GlcNAc(.beta.1-3)Gal(.beta.1-4)Glc(.beta.1-)-
-R [1]
Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-6)]GlcNAc(.beta.1-3)Gal(.beta.1-4)G-
lc(.beta.1-)-R AG223
Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-6)]GlcNAc(.beta.1-3)Gal(.beta.-
1-4)Glc(.beta.1-)- R [9] FH-2
Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R [2]
HSO3(-6)Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R
73-30
Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R [2]
HSO3(-6)Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R
LeuM1
Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R [2]
HSO3(-6)Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R
SU59
HSO3(-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)Gal(.beta.1-)-
-R [2] Group 4. Binds sulfo, sialic acid and Fuc G152
Neu5Ac(.alpha.2-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-6)]GlcNAc(.be-
ta.1- 3)Gal(.beta.1-)-R [2], [5] G72
Neu5Ac(.alpha.2-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-6)]GlcNAc(.bet-
a.1- 3)Gal(.beta.1-)-R [2], [2] G2706
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-
6)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R [2] G27011
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-
6)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R [2] G27037
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-
6)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R [2] G27039
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-
6)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R [2] 2F3
Neu5Ac(.alpha.2-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)Gal(-
.beta.1-)-R [1]
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-
- 3)Gal(.beta.1-)-R
Neu5Ac(.alpha.2-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-6)]GlcNAc(.beta.1-
- 3)Gal(.beta.1-)-R
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-
6)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R 2H5
Neu5Ac(.alpha.2-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)Gal(-
.beta.1-)-R [1]
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-
- 3)Gal(.beta.1-)-R
Neu5Ac(.alpha.2-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-6)]GlcNAc(.beta.1-
- 3)Gal(.beta.1-)-R
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-
6)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R CSLEX1
Neu5Ac(.alpha.2-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)G-
al(.beta.1-)-R [1]
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-
- 3)Gal(.beta.1-)-R HECA-452
Neu5Ac(.alpha.2-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3-
)Gal(.beta.1-)-R [2]
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-
- 3)Gal(.beta.1-)-R
Neu5Ac(.alpha.2-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-6)]GlcNAc(.beta.1-
- 3)Gal(.beta.1-)-R
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)][HSO3(-
6)]GlcNAc(.beta.1-3)Gal(.beta.1-)-R SNH-3
Neu5Ac(.alpha.2-3)Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-3)Ga-
l(.beta.1-)-R [2]
Neu5Ac(.alpha.2-3)[HSO3(-6)]Gal(.beta.1-4)[Fuc(.alpha.1-3)]GlcNAc(.beta.1-
- 3)Gal(.beta.1-)-R Comments: [1] GlycoEpitope database
www.glyco.is.ritsumei.ac.jp [2] Mitsuoka & al. 1998, JBC 273:
11225 [3] Schakel & al. 2002, Immunity 17: 289 [4] Veerman
& al. 1997, Glycobiology 7: 37 [5] Ohmori & al. 2006, Bllod
107: [6] Tsubokawa & al. 2007, FEBS Journal 274: 1833 [7]
Kannagi & al. 2008, Glycoconj. J.: March [8] Kimura & al.
2007, JBC 282: 32200 [9] Izawa & al. 2000, Cancer Res. 60:
1410
TABLE-US-00006 TABLE 4 Tested MSC suspension incubation strategies
(incubation vessel and suspension techniques). Viability was
determined after 2 h with Trypan blue exclusion. Viability by
Trypan Samples blue staining #1 24-well plate NO SUSPENSION a
Buffer control 2 h >95% b Buffer + 100 mU SAT >95% #2 24-well
plate SUSPENSION EVERY 30 MIN a Buffer control 2 h >95% b Buffer
+ 100 mU SAT >90% #3 15 ml conical tube in cell culture
incubator, cap open, suspension every 30 min a Buffer control 2 h
>95% b Buffer + 100 mU SAT >95% #4 15 ml conical tube in 37
C. water bath, cap closed, tapping every 30 min a Buffer control 2
h >90% b Buffer + 100 mU SAT >90% #5 15 ml conical tube in 37
C. shaker, 150 rpm, tube tilted a Buffer control 2 h >95% b
Buffer + 100 mU SAT >95%
TABLE-US-00007 TABLE 5 Flow cytometry with conjugated MAA and MAL-1
plant lectins and UCBMSC 391P cells in different vessels with and
without enforced .alpha.2,3-sialylation. Reaction numbering as in
Table 4. A conical tube in cell incubator (#3) seems to be the best
vessel for enzymatic modifications of MSCs according to increase in
MAA and MAL-1 high populations. Evident changes in cell surface
.alpha.2,3-sialylation is seen in only buffer (.alpha.MEM + 0.5%
human serum albumin) incubated (no enzyme) cells in hypoxic
conditions (conditions #4 and #5). Percentages indicated change as
compared to control cells at time point 0. #1 #2 #3 #4 #5
a2,3-SATIII-induced increase in cell surface a2,3-sialylation %
increase after 2 hours MAA.sup.high 9.9 11.7 12.3 4.6 -2.6
MAL1.sup.high 7.3 12.3 8.8 -0.2 -0.7 normoxia hypoxia
Buffer-induced alterations in cell surface a2,3-sialylation %
increase after 2 hours MAA.sup.high 4 5.1 3.5 17.2 11.5
MAL1.sup.high 3.7 6.5 3.3 19.4 9.9 normoxia hypoxia
TABLE-US-00008 TABLE 6 Enzymatic desialylation of UCB MSCs (391P
p6) in .alpha.MEM Glutamax supplemented with A) 0.5% human serum
albumin (HSA) or B) 0.5% bovine serum albumin (BSA). Control 1 =
time point 0, Control 2 = buffer incubated cells (2 h), V. cholera
desialylation for 2 hours. A. Incubation buffer: .alpha.MEM + 0.5%
HSA B. Incubation buffer: .alpha.MEM + 0.5% BSA MAA-FITC SNA-FITC
MAL1-FITC CHO131 MAA-FITC SNA-FITC MAL1-FITC CHO131 Control 1 0.80%
99.40% 7.70% 99.40% Control 1 1.40% 99.20% 3.10% 97.90% Control 2
15.50% 99.10% 19.50% 87.20% Control 2 37.90% 99.10% 42.70% 84.40%
Desialylation 6.10% 39.20% 10.70% 62.60% Desialylation 23.40%
62.30% 29.70% 39.40%
TABLE-US-00009 TABLE 7 List of plant lectins tested for their
usability to validate changes in cell surface sialylation.
Lectin/specificity Manufacturer Description MAA-FITC (.alpha.2,3SA)
Ey laboratories Combination of 2 isoforms of MAA: MAA1 and MAA2. No
glycan array test of binding specificity by CFG (Consortium for
Functional Glycomics). MAA-biotin (.alpha.2,3SA) Ey laboratories
Combination of 2 isoforms of MAA: MAA1 and MAA2. Not tested by CFG.
MAL1-FITC (.alpha.2,3SA) Vector laboratories, #FL-1311 Specificity
tested (CFG). MAL1 binds to .alpha.2,3-sialylated and sulfated
structures, prefering SA.alpha.2,3Gal.beta.1,4GlcNAc. In addition
MAL1 recongize Neu5Aca2-6Lac and GT3 glycan with chain of 3 sialic
acids. MAL1-FITC has also low binding affinity to non- sialylated
and non-suflated LacNAc structures. MAL1-biotin (.alpha.2,3SA)
Vector laboratories, #B-1315 See above the binding pattern for
MAL1. MAL2-biotin (.alpha.2,3SA) Vector laboratories, #B-1265
Spesificity tested by CFG. MAL2 binds to various sulphated glycan
structures which may or may not carry sialic acids. Preference
towards SA.alpha.2,3Gal.beta.1,3GalNAc has been suggested (Konami
Y. FEBS Lett. 1994). SNA-FITC (.alpha.2,6SA) Vector laboratories
The specificity of FITC conjugated SNA from Vector laboratories has
not been tested by CFG, but SNA-biotin from the same manufacturer
shows high specificity towards .alpha.2,6SA. LFA-FITC (SA,
independent EY laboratories, #F5101-1 No tested binding specificity
of the linkage) by CFG. LFA-biotin (SA, independed of EY
laboratories, #BA-5101-1 No tested binding specificity the linkage)
by CFG. ECA-biotin (terminal Gal) Vector laboratories Recognize
terminal galactose and possibly fucosylated O- glygans. Only the
binding specificity of ECA-FITC (EY laboratories) has been tested
by CFG.
TABLE-US-00010 TABLE 8 List of used glycoform-specific antibodies
for Lewis X (Lex) and sialyl Lewis X (sLex) glycostructures to
validate changes in cell surface sialylation and fucosylation.
Antibody/specificity Manufacturer Description CHO-131, PSGL-1
R&D systems SA(a3)Gal(b4)[Fuc(a3)]GlcNAc sLex on core II
#MAB996 O-glycans/sLex CSLEX, BD Pharmingen
SA(a3)Gal(b4)[Fuc(a3)]GlcNAc CD15s/sLex #551344 TG-1, CD15/Lex
abcam #ab17080 Gal(b4)[Fuc(a3)]GlcNAc HECA-452, BD Pharmingen
SA(a3)Gal(b4)[Fuc(a3)]GlcNAc CLA/sLex #555947
TABLE-US-00011 TABLE 9 Sia-panel expression profile in various
incubation media. The change of positively labelled cells was
compared to freshly labelled cells. 40 min suspension time 0 2 h
suspension incubation incubation Control .alpha.MEM + StemPro +
HBSS + HBSS + Lectin levels .alpha.MEM SAT StemPro SAT HBSS SAT
HBSS SAT MAA 0.90% +7.1 +33 +18.9 +25 +6.9 +22.2 +2.0 +9.8 |MAL-1
0.90% +6.6 +13 +22.1 +11.2 +18.8 +16.5 +1.8 +8.1 SNA 99.50% +0.3
+0.3 +0.1 +0.1 +0.5 +0.2 +0.5 +0.4
TABLE-US-00012 TABLE 10 Validation of .alpha.1,3-fucosylated human
bone marrow-derived mesenchymal stem cells BMMSCs after 1 h FUTVI
enzymatic glycomodification in suspension incubation in .alpha.MEM
+ 0.5% human serum albumin (HSA) buffer. Control cells were
incubated in buffer only without enzyme. CHO-131 BMMSC cell sample
% pos CSLEX HECA 452 TG-1 buffer control 52 3.8 0.8 2.1
a1,3-fucosylation 99.7 97.4 72.6 37.2
TABLE-US-00013 TABLE 11 Number of total amount of cells in cell
culture vessel 6 days after indicated glycomodifications. All cells
were plated after the glycomodification with 1000 cell per
cm.sup.2. Both untreated cells and reaction buffer only incubated
cells served as controls. Reaction number of cells * 10{circumflex
over ( )}6 Neuraminidase 1 h 1,970 Neuraminidase 1 h + SAT 1 h
1,987 SAT 1 h 2,043 incubation control 2,113 untreated cells
1,953
TABLE-US-00014 TABLE 12 Components of .alpha.-MEM culture medium
(Invitrogen Life Science). Molecular Concentration COMPONENTS
Weight (mg/L) mM Amino Acids Glycine 75 50 0.667 L-Alanine 89 25
0.281 L-Alanyl-L-Glutamine 203 406 2 L-Arginine 211 105 0.498
L-Asparagine-H2O 132 50 0.379 L-Aspartic acid 133 30 0.226
L-Cysteine hydrochloride 121 100 0.826 L-Cystine 313 31 0.099
L-Glutamic Acid 147 75 0.51 L-Histidine 155 31 0.2 L-Isoleucine 131
52.4 0.4 L-Leucine 131 52.4 0.4 L-Lysine 146 58 0.397 L-Methionine
149 15 0.101 L-Phenylalanine 165 32 0.194 L-Proline 115 40 0.348
L-Serine 105 25 0.238 L-Threonine 119 48 0.403 L-Tryptophan 204 10
0.049 L-Tyrosine 181 36 0.199 L-Valine 117 46 0.393 Vitamins
Ascorbic Acid 176 50 0.284 Biotin 244 0.1 0.00041 Choline chloride
140 1 0.00714 D-Calcium pantothenate 477 1 0.0021 Folic Acid 441 1
0.00227 Niacinamide 122 1 0.0082 Pyridoxal hydrochloride 204 1
0.0049 Riboflavin 376 0.1 0.000266 Thiamine hydrochloride 337 1
0.00297 Vitamin B12 1355 1.36 0.001 i-Inositol 180 2 0.0111
Inorganic Salts CaCl2--2H2O 147 264 1.8 MgSO4--7H2O 246 200 0.813
KCl 75 400 5.33 NaHCO3 84 2200 26.19 NaCl 58 6800 117.24
NaH2PO4--2H2O 156 158 1.01 Other Components D-Glucose (Dextrose)
180 1000 5.56 Lipoic Acid 206 0.2 0.000971 Phenol Red 376.4 10
0.0266 Sodium Pyruvate 110 110 1
* * * * *
References