U.S. patent application number 15/321679 was filed with the patent office on 2017-07-20 for method for evaluating quality of human mesenchymal stem cell, and monoclonal antibody for use in said method.
This patent application is currently assigned to PUREC CO., LTD.. The applicant listed for this patent is PUREC CO., LTD.. Invention is credited to Yumi IYOKU, Yo MABUCHI, Hideyuki OKANO.
Application Number | 20170204374 15/321679 |
Document ID | / |
Family ID | 55217690 |
Filed Date | 2017-07-20 |
United States Patent
Application |
20170204374 |
Kind Code |
A1 |
IYOKU; Yumi ; et
al. |
July 20, 2017 |
METHOD FOR EVALUATING QUALITY OF HUMAN MESENCHYMAL STEM CELL, AND
MONOCLONAL ANTIBODY FOR USE IN SAID METHOD
Abstract
The present invention aims to obtain a method for quality
evaluation of human mesenchymal stem cells, a method for isolation,
selection and culture of human mesenchymal stem cells, a cell
population of rapidly proliferating human mesenchymal stem cells,
as well as monoclonal antibodies that specifically recognize
rapidly proliferating human mesenchymal stem cells. From a cell
population containing human mesenchymal stem cells, rapidly
proliferating human mesenchymal stem cells are isolated, selected
and cultured. The abundance ratio of cells expressing Ror2 or Fzd5
in the cell population thus isolated, selected and cultured is
quantified to determine whether or not each cell population is
acceptable.
Inventors: |
IYOKU; Yumi; (Tokyo, JP)
; OKANO; Hideyuki; (Tokyo, JP) ; MABUCHI; Yo;
(Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PUREC CO., LTD. |
Izumo-shi, Shimane |
|
JP |
|
|
Assignee: |
PUREC CO., LTD.
Izumo-shi, Shimane
JP
|
Family ID: |
55217690 |
Appl. No.: |
15/321679 |
Filed: |
July 31, 2015 |
PCT Filed: |
July 31, 2015 |
PCT NO: |
PCT/JP2015/071770 |
371 Date: |
December 22, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/04 20130101; C12P
1/00 20130101; C07K 16/28 20130101; G01N 2333/912 20130101; C12N
5/0663 20130101; C12Q 1/6881 20130101; G01N 2333/705 20130101; G01N
33/56966 20130101; C12Y 207/10001 20130101; C12N 15/02
20130101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775; C12Q 1/68 20060101 C12Q001/68; G01N 33/569 20060101
G01N033/569 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 1, 2014 |
JP |
2014-157367 |
Claims
1-20. (canceled)
21. A method for quality evaluation of human mesenchymal stem
cells, which comprises: a step where from a cell population
containing human mesenchymal stem cells, rapidly proliferating
human mesenchymal stem cells are isolated, selected and cultured;
and a step where the abundance ratio of cells expressing Ror2 in
the cell population thus isolated, selected and cultured is
quantified to determine whether or not each cell population is
acceptable.
22. The method for quality evaluation of human mesenchymal stem
cells according to claim 21, wherein the cells expressing Ror2 are
quantified by using anti-Ror2 monoclonal antibody.
23. The method for quality evaluation of human mesenchymal stem
cells according to claim 22, wherein the cells expressing mRNA of
Ror2 are quantified by quantitative PCR.
24. The method for quality evaluation of human mesenchymal stem
cells according to claim 22, wherein the cells expressing Ror2 are
quantified by immunostaining.
25. A method for isolation, selection and culture of human
mesenchymal stem cells, which comprises: a step where from a cell
population containing human mesenchymal stem cells, rapidly
proliferating human mesenchymal stem cells are isolated, selected
and cultured; and a step where the abundance ratio of cells
expressing Ror2 in the cell population thus isolated, selected and
cultured is quantified to determine whether or not each cell
population is acceptable, thus selecting only the cell
population(s) determined to be acceptable.
26. The method for isolation, selection and culture of human
mesenchymal stem cells according to claim 25, wherein the cells
expressing Ror2 are quantified by using anti-Ror2 monoclonal
antibody.
27. The method for isolation, selection and culture of human
mesenchymal stem cells according to claim 26, wherein the cells
expressing mRNA of Ror2 are quantified by quantitative PCR.
28. The method for isolation, selection and culture of human
mesenchymal stem cells according to claim 25, wherein the step
where rapidly proliferating human mesenchymal stem cells are
isolated, selected and cultured comprises a step where the cell
population containing human mesenchymal stem cells is analyzed by
flow cytometry (hereinafter referred to as "FCM") for cells stained
simultaneously with anti-LNGFR monoclonal antibody and anti-Thy1
monoclonal antibody to thereby effect cell sorting of LNGFR.sup.+
Thy1.sup.+ cells.
29. The method for isolation, selection and culture of human
mesenchymal stem cells according to claim 25, wherein the step
where rapidly proliferating human mesenchymal stem cells are
isolated, selected and cultured comprises a step where the cell
population containing human mesenchymal stem cells is analyzed by
FCM for cells stained with anti-Ror2 monoclonal antibody to thereby
effect cell sorting of Ror2.sup.+ cells.
30. The method for isolation, selection and culture of human
mesenchymal stem cells according to claim 28 or 29, which comprises
a step where the cell population is directly prepared from cells
derived from bone marrow or other tissues.
31. The method for isolation, selection and culture of human
mesenchymal stem cells according to claim 30, which comprises a
step where the cell population is prepared by adherent culture of
cells derived from bone marrow or other tissues.
32. The method for isolation, selection and culture of human
mesenchymal stem cells according to claim 28 or 29, wherein the
cell sorting step comprises a step where the positive cells are
seeded in wells of a culture plate and the cells in each well
reaching confluence upon culture are then isolated and
selected.
33. A cell population of rapidly proliferating human mesenchymal
stem cells, which is obtained by: a step where a cell population
containing human mesenchymal stem cells is analyzed by FCM for
cells stained with anti-Ror2 monoclonal antibody to thereby effect
cell sorting of Ror2.sup.+ cells, and these positive cells are
seeded in wells of a culture plate and the cells in each well
reaching confluence upon culture are isolated and selected; and a
step where the abundance ratio of cells expressing Ror2 in the cell
population thus isolated and selected is quantified to determine
whether or not each cell population is acceptable, thus selecting
only the cell population(s) determined to be acceptable.
34. A cell population of rapidly proliferating human mesenchymal
stem cells, which is obtained by: a step where a cell population
containing human mesenchymal stem cells is analyzed by FCM for
cells stained with anti-Ror2 monoclonal antibody to thereby effect
cell sorting of Ror2.sup.+ cells, and these positive cells are
seeded in wells of a culture plate and the cells in each well
reaching confluence upon culture are isolated and selected; and a
step where the abundance ratio of cells expressing Fzd5 in the cell
population thus isolated and selected is quantified to determine
whether or not each cell population is acceptable, thus selecting
only the cell population(s) determined to be acceptable.
35. The cell population of rapidly proliferating human mesenchymal
stem cells according to claim 33 or 34, wherein cells whose
abundance ratio is 63% or higher are determined to be acceptable.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for quality
evaluation of human mesenchymal stem cells, a method for isolation,
selection and culture of human mesenchymal stem cells, a cell
population of rapidly proliferating human mesenchymal stem cells,
as well as monoclonal antibodies that specifically recognize
rapidly proliferating human mesenchymal stem cells.
BACKGROUND ART
[0002] Mesenchymal stem cells (MSCs) are a kind of somatic stem
cells which are increasingly used for clinical applications, after
hematopoietic stem cells, because they have fewer ethical problems
associated with cell collection and have differentiation potency
into various types of tissues such as bone, cartilage, fat, etc.
Mesenchymal stem cells can be isolated through relatively simple
manipulations as described later, and therefore are widely used as
materials for biomaterials, for example, by being induced to
differentiate into cartilage, bone and others mainly in test tubes
and then used for local transplantation.
[0003] As a method for isolation and culture of human mesenchymal
stem cells, the culture method reported in Non-patent Document 1 is
commonly used. However, a cell population obtained by such a
conventional method contains many contaminant cells of less quality
(which have lost their differentiation, proliferation and migration
potency), and these contaminant cells serve as a factor causing
further loss of quality because they affect the cells which should
have inherently had potential.
[0004] Under these conventional circumstances, there has been
established a method for isolation and culture of human mesenchymal
stem cells, which achieves higher proliferation potency,
differentiation potency and migration potency than those in
conventional methods (Non-patent Documents 2 and 3 and Patent
Document 1). According to these Non-patent Documents 2 and 3 and
Patent Document 1, antibodies against CD271 (LNGFR) and CD90 (Thy1)
are used to select LNGFR.sup.+ Thy1.sup.+ cells from human bone
marrow, placental chorion, fat tissue, peripheral blood, dental
pulp and so on, whereby human mesenchymal stem cells can be
enriched.
[0005] Moreover, the selected LINGFR.sup.+ Thy1.sup.+ cells are
subjected to single cell (clone) culture to select a rapidly
expanding lot (REC: Rapidly Expanding Clone), whereby human
mesenchymal stem cells excellent in proliferation potency,
differentiation potency and migration potency can be obtained in
high purity.
[0006] The high purity human mesenchymal stem cells (RECs) thus
obtained were found to have proliferation potency, differentiation
potency and migration potency which were all 1000-fold or more
higher than those of mesenchymal stem cells obtained by
conventional methods.
[0007] According to the features of the above method for isolation
and culture of human mesenchymal stem cells, single cell culture
allows the formation of conditions free from contaminant cells and
thus enables expansion culture while maintaining cell quality. In
particular, because of retaining migration potency, the resulting
cells can be administered via the intravenous route, and therefore
can be expected for use in serious systemic diseases such as bone
and cartilage hypoplasia.
PRIOR ART DOCUMENTS
Patent Documents
[0008] Patent Document 1: JP 2009-60840 A
Non-Patent Documents
[0008] [0009] Non-patent Document 1: Pittenger, M. F., Mackay, A.
M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D.,
Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R.
(1999). Multilineage potential of adult human mesenchymal stem
cells. Science 284, 143-147. [0010] Non-patent Document 2: Mabuchi
Y, Morikawa S, Harada S, Niibe K, Suzuki S, Renault-Mihara F,
Houlihan D D, Akazawa C, Okano H, Matsuzaki Y. (2013).
LNGFR+THY-1+VCAM-1hi+ Cells Reveal Functionally Distinct
Subpopulations in Mesenchymal Stem Cells. Stem Cell Reports 1,
152-165. [0011] Non-patent Document 3: CGH array data, Gene
Expression Omnibus (GEO) (accession number: GSE34484)
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0012] Unlike immortalized cell lines, even RECs cannot avoid the
loss of cell quality when repeatedly subcultured over a long period
of time. However, at present, there is no accurate indicator for
the loss of quality.
[0013] The present invention aims to provide a method for quality
evaluation of human mesenchymal stem cells, a method for isolation,
selection and culture of human mesenchymal stem cells, a cell
population of rapidly proliferating human mesenchymal stem cells,
as well as monoclonal antibodies that specifically recognize
rapidly proliferating human mesenchymal stem cells.
[0014] In addition, gene expression analysis was performed between
RECs and the other clones of less quality (MEC: Moderately
Expanding Clone, SEC: Slowly Expanding Clone) to thereby select
REC-specific genes. The present invention further aims to prepare
novel monoclonal antibodies that recognize proteins expressed from
the specific genes.
Means to Solve the Problem
[0015] To achieve the above aim, the method for quality evaluation
of human mesenchymal stem cells according to the present invention
is characterized in that from a cell population containing human
mesenchymal stem cells, rapidly proliferating human mesenchymal
stem cells are isolated, selected and cultured, and cells
expressing Ror2 or Fzd5 (or the abundance ratio thereof) in the
cell population thus isolated, selected and cultured are quantified
to determine whether or not each cell population is acceptable.
According to this constitution, Ror2 or Fzd5 when expressed alone
allows determination of whether or not a cell population is
composed of RECs, and the determination can be made in a simpler
manner because cultured cells may also be used for this purpose. It
should be noted that LNGFR is not expressed in cultured cells even
when they are RECs, and Thy1 when expressed alone does not allow
determination of whether or not a cell population is composed of
RECs.
[0016] In the above constitution, cells expressing Ror2 or Fzd5 may
be quantified by using anti-Ror2 monoclonal antibody or anti-Fzd5
monoclonal antibody. In this case, mRNA expression of Ror2 may be
quantified by quantitative PCR, or alternatively, cells expressing
Ror2 or Fzd5 may be quantified by immunostaining. However, it
should be noted that Ror2 is expressed extracellularly and hence
analyzed by flow cytometry (hereinafter referred to as "FCM") is
easily applicable for the above purpose, whereas Fzd5 is expressed
intracellularly and visual evaluation such as immunostaining is
therefore suitable for the above purpose.
[0017] On the other hand, to achieve the above aim, the method for
isolation, selection and culture of human mesenchymal stem cells is
characterized in that from a cell population containing human
mesenchymal stem cells, rapidly proliferating human mesenchymal
stem cells are isolated, selected and cultured, and cells
expressing Ror2 or Fzd5 (or the abundance ratio thereof) in the
cell population thus isolated, selected and cultured are quantified
to determine whether or not each cell population is acceptable,
thus selecting only the cell population(s) determined to be
acceptable.
[0018] In this constitution, cells expressing Ror2 or Fzd5 may be
quantified by using anti-Ror2 monoclonal antibody or anti-Fzd5
monoclonal antibody. In this case, cells expressing mRNA of Ror2
may be quantified by quantitative PCR, or alternatively, cells
expressing Ror2 or Fzd5 may be quantified by immunostaining.
[0019] Moreover, the above step where rapidly proliferating human
mesenchymal stem cells are isolated, selected and cultured may
comprise a step where the cell population containing human
mesenchymal stem cells is analyzed by FCM for cells stained
simultaneously with anti-LNGFR monoclonal antibody and anti-Thy1
monoclonal antibody to thereby effect cell sorting of LNGFR.sup.+
Thy1+ cells. Without being limited to this embodiment, the above
step where rapidly proliferating human mesenchymal stem cells are
isolated, selected and cultured may alternatively comprise a step
where the cell population containing human mesenchymal stem cells
is analyzed by FCM for cells stained with anti-Ror2 monoclonal
antibody to thereby effect cell sorting of Ror2.sup.+ cells.
[0020] Further, the above methods may each comprise a step where
the above cell population is directly prepared from cells derived
from each tissue, including bone marrow. On the other hand, the
method comprising the step of effecting cell sorting of Ror2.sup.+
cells may comprise a step where the above cell population is
prepared by adherent culture of cells derived from each tissue,
including bone marrow. This is because cultured cells of RECs are
not positive for LNGFR but are positive for Ror2.
[0021] In the above methods, the above cell sorting step may
comprise a step where the positive cells are seeded in wells of a
culture plate and the cells in each well reaching confluence upon
culture are then isolated and selected.
[0022] To achieve the above aim, the cell population of rapidly
proliferating human mesenchymal stem cells is characterized in that
from a cell population containing human mesenchymal stem cells,
rapidly proliferating human mesenchymal stem cells are isolated,
selected and cultured, and cells expressing Ror2 or Fzd5 (or the
abundance ratio thereof) in the cell population thus isolated,
selected and cultured are quantified to determine whether or not
each cell population is acceptable, thus selecting only the cell
population(s) determined to be acceptable.
[0023] In this characteristic, the cell population containing human
mesenchymal stem cells may be analyzed by FCM for cells stained
simultaneously with anti-LNGFR monoclonal antibody and anti-Thy1
monoclonal antibody to thereby effect cell sorting of LNGFR.sup.+
Thy1+ cells, and these dual positive cells may be seeded in wells
of a culture plate and the cells in each well reaching confluence
upon culture may be isolated and selected, prior to the above
quantification.
[0024] Alternatively, in this characteristic, the cell population
containing human mesenchymal stem cells may be analyzed by FCM for
cells stained with anti-Ror2 monoclonal antibody to thereby effect
cell sorting of Ror2.sup.+ cells, and these positive cells may be
seeded in wells of a culture plate and the cells in each well
reaching confluence upon culture may be isolated and selected,
prior to the above quantification.
[0025] To achieve the above aim of the present invention, a novel
monoclonal antibody according to the present invention is anti-Ror2
monoclonal antibody whose clone name is 7C9. In addition, another
novel monoclonal antibody according to the present invention is
anti-Fzd5 monoclonal antibody whose clone name is 6F5.
Effects of the Invention
[0026] Proteins encoded by the identified two genes (Fzd5 and Ror2:
their details will be described later) are expressed specifically
in RECs, and their expression is not observed in cell populations
of less quality. Moreover, these genes are essential for
maintaining the undifferentiated state of RECs, and they serve as
effective indicators which are closely related to cell functions
and ensure cell performance, but not serve as mere biomarkers, in
light of the following results: 1) inhibition of their expression
includes loss of cell performance; and 2) forced expression of
these genes allows prolonged undifferentiated state, etc.
[0027] As described above, the present invention enables the
provision of a method for quality evaluation of human mesenchymal
stem cells, a method for isolation, selection and culture of human
mesenchymal stem cells, a cell population of rapidly proliferating
human mesenchymal stem cells, as well as monoclonal antibodies that
specifically recognize rapidly proliferating human mesenchymal stem
cells.
[0028] Other aims, constitutions and effects of the present
invention will become apparent from the embodiments of the
invention shown below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows the steps of selection, isolation, culture and
quality evaluation of high quality mesenchymal stem cells
(RECs).
[0030] FIG. 2 shows the results compared for various parameters
among RECs, MECs and SECs.
[0031] FIG. 3 shows figures and graphs which indicate that Ror2 and
Fzd5 are expressed specifically in RECs.
[0032] FIG. 4 shows figures and graphs which indicate that
inhibition of Fzd5 induces loss of cell properties in RECs.
[0033] FIG. 5 shows photographs and graphs which indicate that
forced expression of Fzd5 includes the undifferentiated nature of
RECs.
[0034] FIG. 6 shows photographs and graphs which indicate the
stainability of newly prepared anti-Fzd5 monoclonal antibody.
[0035] FIG. 7 shows photographs and figures which indicate the
stainability of newly prepared anti-Ror2 monoclonal antibody.
[0036] FIG. 8 shows a schematic diagram for quality evaluation of
cultured MSCs using REC-specific antibody.
DESCRIPTION OF EMBODIMENTS
[0037] While referring to the drawings, the steps of selection,
isolation and culture of RECs will be outlined below, followed by
explanations about the purpose and details of each step. In the
present invention, RECs are selected, isolated and cultured in Step
1, and the cultured RECs are evaluated in Step 2. Some exemplary
combinations of these steps are listed in Table 1. All combinations
except for those expressed as "Unable" in the column "Process
evaluation" are able to be implemented. First, the combinations
under Process Nos. P1 and P2 will be described below.
TABLE-US-00001 TABLE 1 Pro- Step 2 Process cess Step 1 Cell eval-
No. Cell source FCM Sorting Culture Well treatment evaluation
uation Comment P1 Tissue (e.g., LNGFR.sup.+ Single Culture for
Selection of Ror2.sup.+ Very Evaluation is possible while ensuring
cell bone marrow) Thy1.sup.+ 2 weeks confluent wells good
performance P2 Tissue (e.g., LNGFR.sup.+ Single Culture for
Selection of Fzd5.sup.+ Good Fzd5 is expressed intracellularly and
hence bone marrow) Thy1.sup.+ 2 weeks confluent wells difficult to
determine by FCM, but can be evaluated by immunostaining or the
like while ensuring cell functions P3 Tissue (e.g., LNGFR.sup.+
Single Culture for Selection of Thy1.sup.+ Unable THY1 is also
expressed in MECs and SECs bone marrow) Thy1.sup.+ 2 weeks
confluent wells and hence cannot be used for evaluation P4 Tissue
(e.g., LNGFR.sup.+ Single Culture for Selection of LNGFR.sup.+
Unable LNGFR is not expressed in cultured cells bone marrow)
Thy1.sup.+ 2 weeks confluent wells P5 Tissue (e.g., LNGFR.sup.+
Multiple Culture for Selection of Ror2.sup.+ Less The proportion of
cells other than RECs can bone marrow) Thy1.sup.+ 2 weeks confluent
wells good be quantified, but purity is low P6 Tissue (e.g.,
Ror2.sup.+ Single Culture for Selection of Ror2.sup.+ Good Sorting
using Ror2 as an indicator results in a bone marrow) 2 weeks
confluent wells lower enrichment rate when compared to the
LNGFR.sup.+ Thy1.sup.+ case P7 Tissue (e.g., Ror2.sup.+ Single
Culture for Selection of Fzd5.sup.+ Good Fzd5 is expressed
intracellularly and hence bone marrow) 2 weeks confluent wells
difficult to determine by FCM, but can be evaluated by
immunostaining or the like while ensuring cell functions P8 Tissue
(e.g., Fzd5.sup.+ Single Culture for Selection of Ror2.sup.+ Unable
Fzd5 is expressed intracellularly and hence bone marrow) 2 weeks
confluent wells difficult to detect by FCM, and is therefore
difficult to use as a marker for cell isolation P9 Tissue (e.g.,
Ror2.sup.+ Multiple Culture for Selection of Ror2.sup.+ Less The
proportion of cells other than RECs can bone marrow) 2 weeks
confluent wells good be quantified, but purity is low P10 Adherent
LNGFR.sup.+ Single Culture for Selection of Ror2.sup.+ Unable LNGFR
is not expressed in cultured cells, cultured cells Thy1.sup.+ 2
weeks confluent wells while THY1 is also expressed in cells other
than RECs P11 Adherent Ror2.sup.+ Single Culture for Selection of
Ror2.sup.+ Good Evaluation is possible while ensuring cell cultured
cells 2 weeks confluent wells performance P12 Adherent Ror2.sup.+
Single Culture for Selection of Fzd5.sup.+ Good Fzd5 is expressed
intracellularly and hence cultured cells 2 weeks confluent wells
difficult to determine by FCM, but can be evaluated by
immunostaining or the like while ensuring cell functions P13
Adherent Fzd5.sup.+ Single Culture for Selection of Ror2.sup.+
Unable Fzd5 is expressed intracellularly and hence cultured cells 2
weeks confluent wells difficult to detect by FCM, and is therefore
difficult to use as a marker for cell isolation P14 Adherent
Ror2.sup.+ Multiple Culture for Selection of Ror2.sup.+ Less The
proportion of cells other than RECs can be cultured cells 2 weeks
confluent wells good quantified, but purity is low P15 Adherent
Ror2.sup.+ Single -- -- -- Good Used without cell evaluation
cultured cells
[0038] [Step 1] FIG. 1 illustrates the step of REC isolation by the
single clone culture method.
1) Mononuclear cells are prepared from human bone marrow (or fat or
placental chorion) and these bone marrow mononuclear cells are
stained with anti-LNGFR and anti-Thy1 (LNGFR.sup.+ Thy1.sup.+ cells
are referred to as LT cells). 2) Flow cytometry (FCM, cell sorter)
is used to effect clone sorting of LNGFR.sup.+ Thy1.sup.+ cells in
a 96-well culture plate (i.e., one cell is seeded per well;
expressed as "Single" in Table 1).
[0039] It should be noted that anti-CD106 monoclonal antibody may
be added to effect clone sorting of LNGFR.sup.+ Thy1.sup.+ cells
that are also strongly positive for CD106.
3) After single cell culture for 2 weeks, the culture plate was
observed under a microscope to select wells reaching confluence,
and the cells contained in each of these wells are determined to be
RECs (Rapidly Expanding Cells). Wells showing delayed cell
expansion, i.e., MECs/SECs (Moderately/Slowly Expanding Cells) are
discarded. 4) The cells contained in the wells selected as RECs are
collected separately on a well-by-well basis. RECs collected from
one well are defined as one lot.
[0040] [Step 2] FIG. 1 further shows the evaluation of cultured
cells using REC markers (anti-Ror2 and anti-Fzd5).
1) The RECs collected from the 96-well plate are transferred to
culture dishes or culture flasks on a well-by-well basis, and
cultured to reach confluence (expansion culture). 2) After
expansion culture, the cells grown in adherent state are collected
from all lots, and an aliquot (about 1 to 3.times.10.sup.3 cells)
of each lot is sampled and single-stained with a monoclonal
antibody against each REC marker (anti-Ror2 or anti-Fzd5). 3) REC
marker-positive cells are analyzed by flow cytometry to determine
the ratio of the REC marker-positive cells in the collected cells
(mRNA expression of Ror2 may be quantified by quantitative PCR, or
alternatively, this ratio may be determined manually under a
microscope). 4) If the above positive ratio is equal to or greater
than a given value (e.g., 65%), such a lot (cell population) is
determined to be acceptable. 5) Cells in the acceptable lots are
filled into freezing vials and stored in liquid nitrogen. 6) These
frozen cells are defined as high quality human mesenchymal stem
cells (product). 7) After the cells in each vial are thawed and
then expanded on a culture dish or flask, a user is finally able to
use at least 1.times.10.sup.10 high purity mesenchymal stem cells
in a stable manner.
[0041] In the above steps, clone sorting of LT cells may be
replaced with clone sorting of Ror2.sup.+ cells (P6 and P7 in Table
1). Moreover, LT cells or Ror2.sup.+ cells may be selected and
seeded in groups of two or more per well of a 96-well culture plate
(expressed as "Multiple" in Table 1; P5, P9 and P14). In this case,
however, the purity is lower than that in clone sorting. It should
be noted that the term "confluence" or "confluent" refers to a
state where 90% or more of the culture vessel surface is coated
with cultured cells. Likewise, the term "semi-confluence" or
"semi-confluent" refers to a state where 70% to 80% of the culture
vessel surface is coated with cultured cells. The size and type of
culture devices to be used may be changed as appropriate depending
on the growth rate of cells.
[0042] In the above embodiment, the above cell population is
directly prepared from cells derived from each tissue, including
bone marrow. However, in the case of sorting for Ror2.sup.+ cells,
the above cell population may be prepared by adherent culture of
cells derived from each tissue, including bone marrow (P10 to P15
in Table 1; expressed as "Adherent cultured cells"). In this case,
bone marrow mononuclear cells are seeded on a medium supplemented
with 10% to 20% serum and bFGF (at 37.degree. C. under 1% to 5%
CO.sub.2) and cultured for about 2 weeks to collect fibroblast-like
adherent cells (CFU-F) appearing after culture. The step of
preparing a cell population may comprise treatment of bone marrow
with collagenase. Alternatively, this step may be designed such
that a cell population is prepared from peripheral blood after
G-CSF administration.
[0043] It should be noted that the evaluation prior to shipment
(Step 2-2) and Step 2-3)) is not always necessary, and adherent
cultured cells may be used and subjected to FCM sorting to isolate
Ror2.sup.+ cells, optionally followed by expansion culture, and the
thus obtained cells may be provided for treatment in Step 2-5) and
the subsequent steps prior to shipment, as in Process P15 in Table
1.
[0044] While referring to FIG. 2, a comparison will now be made of
cell performance between RECs and MECs/SECs. FIG. 2 shows the
results obtained when RECs, MECs and SECs were compared for their
cell performance using various parameter data.
[0045] FIG. 2A shows the results obtained when human bone marrow
mononuclear cells were stained with antibodies against LNGFR and
Thy1, followed by FCM analysis. The area within the ellipse
represents LT cells.
[0046] FIG. 2B is a schematic diagram for single cell isolation
(clone sorting) of LT cells in a 96-well plate.
[0047] FIG. 2C is a graph showing the results measured for cell
counts after single cell culture at fixed time intervals. RECs show
a higher proliferation rate than MECs/SECs, and their count reaches
0.5 to 1.times.10.sup.4 within about 2 weeks. A cell count of 0.5
to 1.times.10.sup.4 is required to establish confluence in a well
of a 96-well plate.
[0048] FIG. 2D shows the results obtained when RECs, MECs and SECs
were induced to differentiate into bone and fat, followed by
quantitative PCR to analyze gene expression specific to bone and
fat cells. RECs were found to be particularly high in
differentiation potency into fat cells when compared to MECs and
SECs.
[0049] FIG. 2E is a graph showing the results obtained when RECs,
MECs and SECs were seeded again in a 96-well plate by clone sorting
and wells showing secondary colony formation were then counted and
compared. Secondary colony formation serves as an indicator for
self-replication potency which is indicative of the
undifferentiated state. About 33% of RECs show secondary colony
formation, whereas only a few colonies are formed from
MECs/SECs.
[0050] FIG. 2F shows the results obtained when the following cell
populations, each having been transformed with a Luc (luciferase)
gene expression vector, i.e., WBMs (MSCs obtained in a standard
manner; WBM is an abbreviation for Whole Bone Marrow), RECs and
MECs/SECs, as well as non-luciferase-labeled WBMs prepared as a
negative control group (Luc(-) Cultured MSCs) were each
administered to immunodeficient mice via the intravenous route,
followed by intraperitoneal administration of luciferin serving as
a substrate for Luc to observe luminescence from luciferase by
using an in vitro detection system (IVIS) at 24 hours after
transplantation. The upper panel shows a graph obtained as follows:
Luc luminescence intensity in each mouse was expressed numerically
and, relative to the WBM MSC-transplanted group which was set to
100%, the luminescence ratio (%) was plotted for the mice
transplanted with the other cells. The lower panel shows images of
Luc luminescence in the recipient mice of the respective groups. As
can be seen from these results, the REC-transplanted mice show
extremely low luminescence intensity in their lungs, thus
indicating that RECs are rarely trapped into capillary vessels in
the lungs, whereas MECs/SECs are trapped almost at the same level
as WBMs (cultured MSCs obtained in a standard manner) and remain in
the lungs.
[0051] In view of all the above results, RECs are a cell population
excellent in proliferation potency, differentiation potency and
migration potency, and are particularly advantageous in that they
have migration potency comparable to that of MSCs in fresh bone
marrow, in terms of being able to be administered systemically
against intractable diseases as described later.
[0052] According to the experiments made by the inventors,
including those shown above, RECs are characterized by the
following, when compared to normal MSCs:
[0053] 1. being a morphologically very uniform cell population;
[0054] 2. showing no cellular aging;
[0055] 3. having a high division rate and being able to be cultured
and amplified while retaining the undifferentiated nature;
[0056] 4. begin a cell population easy to differentiate into bone
and fat due to high differentiation potency; and
[0057] 5. retaining migration potency
[0058] RECs are the most undifferentiated cell population among
human MSCs, and have the most similar properties to MSCs in bone
marrow. Moreover, when compared to MECs/SECs or MSCs obtained in a
standard manner, RECs are a fresh and less mutated cell population
ensuring cell performance due to their higher differentiation,
proliferation and migration potency.
[0059] Next, while referring to FIG. 3, an explanation will be made
about the identification of undifferentiated MSC (REC)-specific
genes Ror2 and Fzd5.
[0060] The expression levels of genes expressed in RECs, MECs and
SECs were compared by the DNA array method, thus confirming that
Fzd5, which is one of the Wnt receptors, and its co-receptor Ror2
were both specific to RECs.
[0061] FIG. 3A shows a comparison of Ror2 mRNA expression in RECs,
MECs and SECs, as measured by quantitative PCR.
[0062] FIG. 3B shows a comparison of Fzd5 mRNA expression in RECs,
MECs and SECs, as measured by quantitative PCR.
[0063] FIG. 3C shows a comparison of Fzd5 protein expression, as
measured by Western blotting.
[0064] FIG. 3D shows photographs compared for intracellular
localization of Fzd5 protein, as observed by immunofluorescent
staining.
[0065] As a result of evaluation by the above several analysis
methods, it can be confirmed that the expression of Fzd5 and Ror2
is specific to RECs. Thus, the respective expression of Fzd5 and
Ror2, when detected and quantified, would be effective as an
indicator for cell quality evaluation of RECs. Moreover, the newly
prepared anti-Fzd5 monoclonal antibody and anti-Ror2 monoclonal
antibody are able to detect and quantify their target protein
antigens in any technique selected from flow cytometry, Western
blotting and immunofluorescent staining.
[0066] Next, while referring to FIG. 4, an examination will be made
of cellular aging induction by loss of function of Fzd5.
[0067] The RNA interference method is a technique to examine the
function of a target gene by introduction of short RNA (shRNA)
having a sequence complementary to target mRNA into cells to
thereby disrupt the target mRNA. FIG. 4 shows the results obtained
from a series of experiments on cell properties in RECs when Fzd5
mRNA was disrupted by shRNA having a sequence complementary to Fzd5
(shFZD5), in comparison with the control group (shCTRL; shRNA
having a random sequence not complementary to Fzd5).
[0068] FIG. 4A is a graph showing the mRNA level of Fzd5, as
quantified by quantitative PCR, after introduction of shFZD5 or
shCTRL into RECs. When the mRNA level of Fzd5 in the control group
(shCTRL, short hairpin control) was set to 100, the mRNA level of
Fzd5 was reduced to about 40% in RECs forced to express shFZD5.
[0069] FIG. 4B is a graph whose vertical axis plots cell counts in
the group forced to express shFZD5 after introduction of shFZD5 or
shCTRL into RECs, relative to cell counts in the control group
which are set to 1, and whose horizontal axis plots the number of
days after shRNA introduction. RECs forced to express shFZD5 showed
a sudden reduction in their cell count when compared to the control
group, thus suggesting that Rzd5 inhibition would induce a
reduction in proliferation potency.
[0070] FIG. 4C shows images of fat droplets stained with Oil-Red-O
at 14 days of culture, after introduction of shFZD5 or shCTRL into
RECs and the subsequent induction of their differentiation into fat
cells. Fzd5 inhibition was found to induce a reduction in
differentiation potency into fat cells, when compared to the
control group.
[0071] The activity of SA-.beta.-gal serving as an indicator for
cellular aging can be detected by blue staining upon addition of
its substrate X-gal. FIG. 4D shows images stained by x-gal staining
after introduction of shFZD5 or shCTRL into RECs, along with a
graph which plots the frequency of cells having SA-.beta.-gal
activity in each cell population.
[0072] FIG. 4E is a graph showing the mRNA level of p16(INK4a)
serving as an indicator for cellular aging, as quantified by
quantitative PCR. When the control group was set to 100, the mRNA
level of p16 in shFZD5-receiving RECs was about 300, thus
indicating that inhibition of Fzd5 expression induced cellular
aging.
[0073] FIG. 4F shows images observed for stress fiber formation by
intracellular staining with anti-F-actin antibody in each cell
population after introduction of shFZD5 or shCTRL into RECs, along
with a graph which plots the averaged area (cell size) of
individual cells in these respective cell populations.
[0074] In view of the above results, inhibition of the Fzd5
function in RECs induces a reduction in proliferation potency, a
reduction in differentiation potency, cellular aging, as well as a
reduction in migration potency and an increase in cell size due to
stress fiber formation, thus resulting in the same properties as in
MECs/SECs. This suggests that Fzd5 would be a functional molecule
ensuring the maintenance of cell performance in RECs, but is not a
mere biomarker.
[0075] Next, while referring to FIG. 5, a discussion will be made
about the long-term maintenance of proliferation potency by gain of
function of Fzd5.
[0076] The full-length cDNA of Fzd5 was forced to be expressed in
RECs to cause constitutive expression of Fzd5 mRNA, and the effect
thereof on cell functions was confirmed. For this purpose, an
expression vector is constructed to carry Fzd5 cDNA and a
fluorescent protein GFP (Green Fluorescent Protein) in tandem to
thereby allow co-expression of GFP in Fzd5 gene-receiving cells, so
that the expression of the introduced gene can be confirmed under a
fluorescence microscope.
[0077] FIG. 5A shows photographs observed for morphology of
GFP-expressing cells under a fluorescence microscope. These images
show cell morphology at 28 days after gene transfer in the cell
population transformed with Fzd5 cDNA and GFP (Fzd5) as well as the
control group transformed with the GFP gene alone (CTRL).
[0078] In the control group, there appear many large-sized
multipolar cells which are characteristic of cellular aging, as
indicated with the arrows in the figure, whereas almost all of the
Fzd5-expressing RECs retain their bipolar morphology with small
cytoplasm.
[0079] FIG. 5B is a graph whose vertical axis plots cell counts of
Fzd5-expressing RECs, relative to cell counts in the control group
which are set to 1, and whose horizontal axis plots the number of
days after gene transfer. RECs forced to express Fzd5 were found to
retain their proliferation potency for a long period of time, when
compared to the control group.
[0080] In view of the above results, Fzd5-mediated stimulation of
Wnt signaling would be expected to allow long-term culture
amplification in a state maintaining the undifferentiated
nature.
[0081] Subsequently, while referring to FIG. 6, an explanation will
be made about the preparation of a novel monoclonal antibody
against human Fzd5.
[0082] The extracellular region of a human Fzd5 antigen was used as
an immunogen to immunize host mice and hybridomas were then
prepared in accordance with standard procedures, followed by
screening with Ba/F3 cells engineered to express the Fzd5 gene,
thereby obtaining a novel anti-Fzd5 monoclonal antibody (clone
name: 6F5).
[0083] This antibody was used to confirm whether or not the Fzd5
protein was able to be detected by various techniques.
[0084] Ba/F3 cells forced to express the extracellular region of
Fzd5 were stained with biotin-labeled 6F5 antibody and then
fluorescently labeled with streptavidin (SAV)-PE, followed by flow
cytometry analysis. FIG. 6A shows a histogram whose horizontal axis
plots the fluorescence intensity of PE. The shaded histogram in the
figure represents a negative control where an isotype control was
added as a primary antibody, while the open histogram represents
the PE fluorescence intensity in the sample stained with 6F5. The
range indicated with the horizontal bar in the figure represents
the range of Fzd5.sup.+ cells, while the numerical value represents
the positive rate (%).
[0085] FIG. 6B shows the results of Western blotting obtained when
intracellular proteins were prepared from three different clones of
RECs and the Fzd5 protein was detected using 6F5 as a primary
antibody. For use as a negative control, intracellular proteins
were prepared from the monkey kidney-derived cell line COST.
[0086] FIG. 6C shows images obtained when REC cells were stained
with 6F5-biotin as a primary antibody and then fluorescently
labeled with streptavidin (SAV)-Alexa 555, followed by observation
under a fluorescence microscope. The anti-Fzd5 antibody (6F5) was
found to be available for use in all of flow cytometry, Western
blotting and immunofluorescent staining.
[0087] Next, while referring to FIG. 7, an explanation will be made
about the preparation of a novel monoclonal antibody against a
human Ror2 antigen.
[0088] A human Ror2 antigen was used as an immunogen to newly
prepare an anti-Ror2 antibody (clone name: 7C9).
[0089] FIG. 7A shows the results obtained when RECs were stained
with 7C9-biotin as a primary antibody and then labeled with SAV-PE,
followed by flow cytometry to detect PE fluorescence. A sample
prepared by adding an isotype control antibody as a primary
antibody is used as a negative control. FIG. 7A is a
two-dimensional dot plot whose vertical axis plots FITC
fluorescence (RECs are all negative because they are not stained)
and whose horizontal axis plots PE fluorescence. Compare to the
area that are free from PE fluorescence-emitting cells in the
negative control group (i.e., the area within the trapezoid in the
figure: 0.011%), a Ror2 expressing cell population was found as a
PE fluorescence in the sample stained with clone 7C9 (69.3%).
[0090] FIG. 7B is an image obtained when RECs were immunostained
with 7C9-biotin as a primary antibody and fluorescently labeled
with streptavidin-Alexa 488, and then observed for expression of
the Ror2 protein under a fluorescence microscope. Most of these
RECs were confirmed to express the Ror2 protein.
[0091] FIG. 7C shows the results obtained when fresh bone marrow
cells were triple stained with LNGFR-APC, Thy1-FITC and Ror2-PE
(monoclonal antibodies against their respective antigens), followed
by flow cytometry analysis. The left panel shows a figure whose
vertical axis plots LNGFR expression and whose horizontal axis
plots Thy1 expression, and the boxed area represents a LNGFR.sup.+
Thy1+ cell population containing human MSCs with high frequency.
The right figure shows the LNGFR Thy1+ cell population extracted
alone. The horizontal axis plots FSC serving as an indicator for
cell size, while the vertical axis plots PE fluorescence from
anti-Ror2-biotin antibody (7C9) labeled with SAV-PE. The boxed area
represents the Ror2-positive area determined on the basis of the
negative control, and the numerical value represents the positive
rate (%). In the case of using clone 7C9, 92.3% of LNGFR.sup.+
Thy1+ cells are Ror2-positive; and hence clone 7C9 can be used as a
selection marker for MSCs to replace LNGFR Thy1.
[0092] Using the newly prepared anti-Ror2 monoclonal antibody, the
Ror2 protein can be detected and quantified by flow cytometry and
immunofluorescent staining (FIGS. 7A and 7B).
[0093] Furthermore, the newly prepared anti-Ror2 can also be used
as a marker for MSCs contained in bone marrow (FIG. 7C).
[0094] Next, while referring to FIG. 8, an explanation will be made
about the procedures for cell quality evaluation using anti-Ror2
antibody or anti-Fzd5 antibody.
[0095] Human MSCs cultured in standard adherent culture (or
subcultured RECs) are collected and stained with REC-specific
monoclonal antibody (anti-Ror2 antibody or anti-Fzd5 antibody).
[0096] The frequency (% content) of positive cells is measured by
flow cytometry. Alternatively, the frequency (% content) of
positive cells may be measured under a fluorescence microscope,
instead. These measurements allow quantification of how many RECs
are contained in the cell population, which in turn allows
evaluation of cell quality, i.e., evaluation of what degree of
differentiation, proliferation and migration potency is possessed
by the target MSCs. According to the experiments made by the
inventors, the Ror2-positive rate of RECs was 72%.+-.8.9% averaged
from five lots. Thus, for example, the lowest value, i.e., at least
63% or at least 65% may be used as a reference value for
acceptability determination.
INDUSTRIAL APPLICABILITY
[0097] The present invention enables the provision of a technique
for efficient isolation and culture of human mesenchymal stem cells
available for use in the treatment of systemic diseases, and also
enables quality evaluation on which to determine whether or not the
resulting cell population is suitable for transplantation and/or
exerts efficacy.
[0098] Among newly prepared monoclonal antibodies showing
stainability specific to high purity mesenchymal stem cells,
candidates suitable for cell isolation can be provided as reagents
for isolation of mesenchymal stem cells by being immobilized on
magnetic nanoparticles. Moreover, as reagents for cell evaluation
to test the quality of the isolated mesenchymal stem cells,
fluorescent substance-labeled antibodies or cell staining reagents
can be provided for practical use.
[0099] Mesenchymal stem cells are not only used as materials for
biomaterials, as previously known, but also can be expected to have
various applications by taking advantage of their pluripotency, as
exemplified by administration to myasthenia gravis, chronic
rheumatism and other diseases, as well as co-transplantation as
supporting cells to provide a tissue scaffold (niche) for cell
therapy required for treatment of severe diseases including spinal
cord injury, hart and vascular failure, chronic liver failure and
so on. In particular, the use of RECs retaining their migration
potency would be expected to provide a therapeutic effect never
seen before when applied to metabolic diseases such as systemic
bone and cartilage diseases including hypophosphatasia, for which
no therapy has been found, as well as GVHD and all other diseases
whose treatment requires administration via the intravenous
route.
* * * * *