U.S. patent number 5,192,553 [Application Number 07/269,926] was granted by the patent office on 1993-03-09 for isolation and preservation of fetal and neonatal hematopoietic stem and progenitor cells of the blood and methods of therapeutic use.
This patent grant is currently assigned to Biocyte Corporation. Invention is credited to Edward A. Boyse, Hal E. Broxmeyer, Gordon W. Douglas.
United States Patent |
5,192,553 |
Boyse , et al. |
March 9, 1993 |
**Please see images for:
( Certificate of Correction ) ( Reexamination Certificate
) ** |
Isolation and preservation of fetal and neonatal hematopoietic stem
and progenitor cells of the blood and methods of therapeutic
use
Abstract
The present invention relates to hematopoietic stem and
progenitor cells of neonatal or fetal blood that are cryopreserved,
and the therapeutic uses of such stem and progenitor cells upon
thawing. In particular, the present invention relates to the
therapeutic use of fetal or neonatal stem cells for hematopoietic
(or immune) reconstitution. Hematopoietic reconstitution with the
cells of the invention can be valuable in the treatment or
prevention of various diseases and disorders such as anemias,
malignancies, autoimmune disorders, and various immune dysfunctions
and deficiencies. In another embodiment, fetal or neonatal
hematopoietic stem and progenitor cells which contain a
heterologous gene sequence can be used for hematopoietic
reconstitution in gene therapy. In a preferred embodiment of he
invention, neonatal or fetal blood cells that have been
cryopreserved and thawed can be used for utologous (self)
reconstitution.
Inventors: |
Boyse; Edward A. (Tucson,
AZ), Broxmeyer; Hal E. (Indianapolis, IN), Douglas;
Gordon W. (New York, NY) |
Assignee: |
Biocyte Corporation (New York,
NY)
|
Family
ID: |
26817651 |
Appl.
No.: |
07/269,926 |
Filed: |
November 10, 1988 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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119746 |
Nov 12, 1987 |
5004681 |
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Current U.S.
Class: |
424/529;
424/93.21; 435/2; 435/374; 435/378; 435/455 |
Current CPC
Class: |
C12N
5/0647 (20130101); A61K 35/28 (20130101); A61K
35/50 (20130101); A61K 35/51 (20130101); C12N
2502/11 (20130101); C12N 2502/30 (20130101) |
Current International
Class: |
A61K
35/14 (20060101); C12N 5/06 (20060101); A61K
035/50 (); A61K 035/14 () |
Field of
Search: |
;435/2,172.1,172.3,240.2,240.26 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
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|
Primary Examiner: Rosen; Sam
Parent Case Text
This application is a continuation-in-part of copending U.S.
application Ser. No. 07/119,746 filed Nov. 12, 1987, now U.S. Pat.
No. 5,004,681 which is incorporated by reference herein in its
entirety.
Claims
What is claimed is:
1. A method for obtaining human neonatal for fetal hematopoietic
stem or progenitor cells comprising:
(a) isolating human neonatal or fetal blood components containing
hematopoietic stem or progenitor cells;
(b) cryopreserving the blood components; and
(c) thawing the blood components,
such that the stem or progenitor cells are viable.
2. The method according to claim 1 further comprising the step
after (c) of removing a cryopreservative.
3. The method according to claim 1 further comprising the step of
growing the stem or progenitor cells in vitro.
4. The method according to claim 1 further comprising the step of
enriching for stem and progenitor cells by a cell separation
procedure.
5. The method according to claim 1 in which the blood components
comprise whole blood.
6. The method according to claim 1 or 5 in which the blood
components are isolated by collection from an umbilical cord.
7. The method according to claim 1 or 5 in which the blood
components are isolated by collection from a placenta.
8. The method according to claim 1 or 5 in which the blood
components are isolated by collection from both an umbilical cord
and a placenta of the same individual.
9. The method according to claim 1 in which the cryopreservation is
by use of a cryoprotective agent.
10. The method according to claim 9 in which the cryoprotective
agent comprises dimethyl sulfoxide.
11. The method according to claim 1 in which the cryopreservation
is by use of liquid nitrogen.
12. The method according to claim 9 in which the cryopreservation
further comprises the use of liquid nitrogen.
13. A method for hematopoietic or immune reconstitution of a human
comprising:
(a) isolating human neonatal or fetal blood components containing
hematopoietic stem cells;
(b) cryopreserving the blood components;
(c) thawing the blood components; and
(d) introducing the blood components into a suitable human
host,
such that the hematopoietic stem cells are viable and can
proliferate within the host.
14. The method according to claim 13 in which the stem cells are
autologous to the host.
15. The method according to claim 13 in which the stem cells are
syngeneic to the host.
16. The method according to claim 13 in which the stem cells are
allogeneic to the host.
17. The method according to claim 16 in which the host has
Fanconi's anemia.
18. The method according to claim 13 in which the blood components
comprise whole blood.
19. The method according to claim 13 in which the blood components
are isolated by collection from an umbilical cord.
20. The method according to claim 13 in which the blood components
are isolated by collection from a placenta.
21. The method according to claim 13 in which the host is
immunodeficient.
22. The method according to claim 21 in which the immunodeficiency
is by reason of irradiation.
23. The method according to claim 21 in which the immunodeficiency
is by reason of chemotherapy.
24. The method according to claim 21 in which the immunodeficiency
is by reason of infection by a pathogenic microorganism.
25. The method according to claim 21 in which the host has a
malignant solid tumor.
26. The method according to claim 13 in which the host has
anemia.
27. The method according to claim 26 in which the host has
Fanconi's anemia.
28. The method according to claim 13 in which the host
hyperproliferative stem cell disorder.
29. The method according to claim 13 in which the host has a
hematopoietic malignancy.
30. The method according to claim 29 in which the hematopoietic
malignancy is a leukemia.
31. The method according to claim 29 in which the hematopoietic
malignancy is a lymphoma.
32. The method according to claim 13 in which the host has an
autoimmune disease.
33. The method according to claim 13 in which the host has a
hemolytic disorder.
34. The method according to claim 13 in which the host has a
genetic disorder.
35. The method according to claim 34 in which the genetic disorder
is Fanconi's anemia.
36. The method according to claim 13 which further comprises, after
step (a) or step (c), introducing a heterologous gene sequence into
the stem cells, which gene sequence is stably incorporated and
capable of expression by progeny of the stem cells.
37. The method according to claim 36 in which the host has a
genetic disorder.
38. The method according to claim 37 in which the heterologous gene
sequence comprises a sequence encoding hemoglobin.
39. The method according to claim 37 in which the host has
thalassemia.
40. The method according to claim 37 in which the host has sickle
cell disease.
41. The method according to claim 37 in which the host has
anemia.
42. The method according to claim 36 in which the host is
immunodeficient.
43. The method according to claim 42 in which the immunodeficiency
is by reason of infection by a pathogenic microorganism.
44. The method according to claim 36 in which the host is infected
by a pathogenic microorganism, and in which the heterologous gene
sequence is expressed as a product which is toxic to the pathogenic
microorganism without significant detriment to the host.
45. The method according to claim 36 in which the heterologous gene
sequence is expressed as a nucleic acid sequence that is
complementary to and can hybridize to a nucleic acid of a
pathogenic microorganism.
46. The method according to claim 45 in which the pathogenic
microorganism is Human Immunodeficiency Virus.
47. A method for hematopoietic or immune reconstitution of a human
comprising:
(a) isolating human neonatal or fetal blood components containing
hematopoietic stem and progenitor cells;
(b) cryopreserving the blood components;
(c) thawing the blood components; and
(d) introducing the blood components into a suitable human
host,
such that the hematopoietic stem and progenitor cells are viable
and can proliferate within the host.
48. The method according to claim 47 in which the stem and
progenitor cells are autologous to the host.
49. The method according to claim 47 in which the stem and
progenitor cells are syngeneic to the host.
50. The method according to claim 47 in which the stem and
progenitor cells are allogeneic to the host.
51. The method according to claim 50 in which the host has
Fanconi's anemia.
52. The method according to claim 47 in which the blood components
comprise whole blood.
53. The method according to claim 47 in which the blood components
are isolated by collection from an umbilical cord.
54. The method according to claim 47 in which the blood components
are isolated by collection from a placenta.
55. A method for obtaining cryopreserved human neonatal or fetal
hematopoietic stem cells derived from the blood comprising:
(a) isolating human neonatal or fetal blood components containing
human neonatal or fetal hematopoietic stem cells; and
(b) cryopreserving the blood components, such that the stem cells
remain viable.
56. The method according to claim 55 in which the blood components
comprise whole blood.
57. A method for hematopoietic or immune reconstitution of a human
comprising introducing into the human a composition comprising
human neonatal or fetal hematopoietic stem cells derived from the
blood, in which the stem cells have been previously
cryopreserved.
58. The method according to claim 57 in which the composition
further comprises human neonatal or fetal hematopoietic progenitor
cells derived from the blood, in which the progenitor cells have
been previously cryopreserved.
59. The method according to claim 57 or 58 in which the composition
comprises whole neonatal or fetal blood.
60. A method for hematopoietic or immune reconstitution of a human
comprising introducing into the human a composition comprising
human neonatal or fetal hematopoietic stem cells derived from the
blood, in which the stem cells are progeny of cells which have been
previously cryopreserved.
61. The method according to claim 60 in which the composition
further comprises human neonatal or fetal hematopoietic progenitor
cells derived from the blood, in which the progenitor cells are
progeny of cells which have been previously cryopreserved.
62. The method according to claim 60 or 61 in which the composition
comprises whole neonatal or fetal blood.
63. The method according to claim 57 in which a heterologous gene
sequence is stably incorporated in the same cells.
64. The method according to claim 58 in which a heterologous gene
sequence is stably incorporated in the progenitor cells.
Description
TABLE OF CONTENTS
1. Introduction
2. Background of the Invention
2.1. Hematopoietic Stem and Progenitor Cells
2.2. Reconstitution of the Hematopoietic System
1 2.3. Cryopreservation of Cells
2.4. Gene Therapy
3. Summary of the Invention
3.1. Definitions
4. Description of the Figures
5. Detailed Description of the Invention
5.1. Isolation of Fetal or Neonatal Hematopoietic Stem and
Progenitor Cells
5.1.1. Collection of Neonatal Blood
5.1.1.1. Volume
5.1.1.2. Preferred Aspects
5 1 1.2.1. Collection Kit
5.1.1.2.2. Vaginal Delivery of the Term Infant
5.1.1.2.3. Other Circumstances of Birth and Delivery
5.1.1.2.3.1. Premature Birth
5.1.1.2.3.2. Multiple Births
5.1.1.2.3.3. Caesarian Delivery
5.1.1.2.3.4. Complicated Delivery
5.1.1.2.3.5. Abnormal Placenta
5.1.1.2.3.6. Collection from the Delivered Placenta
5.1.1.2.3.7. Medical Conditions of the Mother
5.1.1.2.3.8. Unplanned Delivery
5.1.1.2.4. Recordation of Data
5.1.2. Inspection and Testing of Neonatal blood
5.1.3. Optional Procedures
5.1.3.1. Enrichment for Hematopoietic Stem and Progenitor Cells:
Cell Separation Procedures
5.1.3.2. In Vitro Cultures of Hematopoietic Stem and Progenitor
Cells
5.2. Cryopreservation
5.3. Recovering Stem and Progenitor Cells from the Frozen State
5.3.1. Thawing
5.3.2. Optional Procedures
5.4. Examination of Cells Recovered for Clinical Therapy
5.4.1. Identity Testing
5.4.2. Assays for Stem and Progenitor Cells
5.5. Hematopoietic Reconstitution
5.6. Therapeutic Uses
5.6.1. Diseases Resulting from a Failure or Dysfunction of Normal
Blood Cell Production and Maturation
5.6.2. Hematopoietic Malignancies
5.6.3. Malignant Solid Tumors of Non-Hematopoietic Origin
5.6.4. Autoimmune Disorders
5.6.5. Gene Therapy
5.6.6. Miscellaneous Disorders Involving Immune Mechanisms
5.7. Generation and Use of Hematopoietic Stem and Progenitor Cell
Progeny
6. Examples
6.1. Collection of Human Umbilical Cord Blood and Placental
Blood
6.2. Hematopoietic Stem and Progenitor Cells in Collected Cord
Blood
6.3. Enrichment for Human Hematopoietic Stem and Progenitor Cells:
Cell Separation Procedures
6.3.1. Density Separations
6.3.2. Adherence/Non-Adherence Separation
6.4. Cryopreservation of Cord Blood Stem and Progenitor Cells
6.5. Cell Thawing
6.6. Human Hematopoietic Stem and Progenitor Cell Assay
6.6.1. CFU-GM Assay
6.6.1.1. Preparation of McCoy's 5A Medium
6.6.1.2. Preparation of Human 5637 Urinary Bladder Carcinoma Cell
Line Conditioned Medium
6.6.1.3. Preparation of Murine Pokeweed Mitogen Spleen Cell
Conditioned Medium
6.6.2. BFU-E-2 and BFU-E-1/CFU-GEMM Assay
6.6.2.1. Preparation of 2.1% Methyl Cellulose
6.6.2.2. Preparation of Hemin
6.6.2.3. Preparation of Iscove's Modified Dulbecco's Medium
6.6.3. Stem Cell Colony Forming Unit Assay
6.6.4. Assay of the Proliferative Status of Stem and Progenitor
Cells
6.7. Recovery After Freeze-Thawing of Human Hematopoietic
Progenitor Cells Derived from Cord Blood
6.8. Calculations of the Reconstituting Potential of Cord Blood
6.9. In Vitro Culture Conditions for Hematopoietic Stem and
Progenitor Cells
6.10. Mouse Dissection Protocols
6.10.1. Bone Marrow Dissection
6.10.2. Spleen Dissection
6.11. Hematopoietic Reconstitution of Adult Mice with Syngeneic
Fetal or Neonatal Stem Cells
6.11.1. Hematopoietic Reconstitution of Lethally-Irradiated Mice
with Stem Cells in Blood of the Near-Term Fetus
6.11.2. Hematopoietic Reconstitution of Mice with a Lesser Volume
of Near-Term Fetal Blood But Not with Adult Blood
6.11.3. Hematopoietic Reconstitution with Blood of Newborn Mice in
Volumes as Low as Ten Microliters
6.11.4. Hematopoietic Reconstitution with Blood of Newborn Mice in
Volumes of 10 or 15 Microliters
6.12. Hematopoietic Reconstitution For Treatment of Fanconi's
Anemia
6.13. Flowchart: Description of a Service
1. INTRODUCTION
The present invention is directed to hematopoietic stem and
progenitor cells of neonatal or fetal blood, that are
cryopreserved, and the therapeutic uses of such stem and progenitor
cells upon thawing. Such cells can be therapeutically valuable for
hematopoietic reconstitution in patients with various diseases and
disorders. In a preferred embodiment, neonatal cells that have been
cryopreserved and thawed, can be used for autologous (self)
hematopoietic reconstitution.
The invention also relates to methods for collection and
cryopreservation of the neonatal and fetal stem and progenitor
cells of the invention.
2. BACKGROUND OF THE INVENTION
2.1. Hematopoietic Stem and Progenitor Cells
The morphologically recognizable and functionally capable cells
circulating in blood include erythrocytes, neutrophilic,
eosinophilic, and basophilic granulocytes, B-, T-, nonB-, non
T-lymphocytes, and platelets. These mature cells derive from and
are replaced, on demand, by morphologically recognizable dividing
precursor cells for the respective lineages such as erythroblasts
for the erythrocyte series, myeloblasts, promyelocytes and
myelocytes for the granulocyte series, and megakaryocytes for the
platelets. The precursor cells derive from more primitive cells
that can simplistically be divided into two major subgroups: stem
cells and progenitor cells (for review, see Broxmeyer, H.E., 1983,
"Colony Assays of Hematopoietic Progenitor Cells and Correlations
to Clinical Situations," CRC Critical Reviews in
Oncology/Hematology 1(3):227-257). The definitions of stem and
progenitor cells are operational and depend on functional, rather
than on morphological, criteria. Stem cells have extensive
self-renewal or self-maintenance capacity (Lajtha, L.G., 1979,
Differentiation 14:23), a necessity since absence or depletion of
these cells could result in the complete depletion of one or more
cell lineages, events that would lead within a short time to
disease and death. Some of the stem cells differentiate upon need,
but some stem cells or their daughter cells produce other stem
cells to maintain the precious pool of these cells. Thus, in
addition to maintaining their own kind, pluripotential stem cells
are capable of differentiation into several sublines of progenitor
cells with more limited self-renewal capacity or no self-renewal
capacity. These progenitor cells ultimately give rise to the
morphologically recognizable precursor cells. The progenitor cells
are capable of proliferating and differentiating along one, or more
than one, of the myeloid differentiation pathways (Lajtha, L.G.
(Rapporteur), 1979, Blood Cells 5:447).
Stem and progenitor cells make up a very small percentage of the
nucleated cells in the bone marrow, spleen, and blood. About ten
times fewer of these cells are present in the spleen relative to
the bone marrow, with even less present in the adult blood. As an
example, approximately one in one thousand nucleated bone marrow
cells is a progenitor cell; stem cells occur at a lower frequency.
These progenitor and stem cells have been detected and assayed for
by placing dispersed suspensions of these cells into irradiated
mice, and noting those cells that seeded to an organ such as the
spleen and which found the environment conducive to proliferation
and differentiation. These cells have also been quantified by
immobilizing the cells outside of the body in culture plates (in
vitro) in a semi-solid support medium such as agar,
methylcellulose, or plasma clot in the presence of culture medium
and certain defined biomolecules or cell populations which produce
and release these molecules. Under the appropriate growth
conditions, the stem or progenitor cells will go through a
catenated sequence of proliferation and differentiation yielding
mature end stage progeny, which thus allows the determination of
the cell type giving rise to the colony. If the colony contains
granulocytes, macrophages, erythrocytes, and megakaryocytes (the
precursors to platelets), then the cell giving rise to them would
have been a pluripotential cell. To determine if these cells have
self-renewal capacities, or stemness, and can thus produce more of
their own kind, cells from these colonies can be replated in vivo
or in vitro. Those colonies, which upon replating into secondary
culture plates, give rise to more colonies containing cells of
multilineages, would have contained cells with some degree of
stemness. The stem cell and progenitor cell compartments are
themselves heterogeneous with varying degrees of self-renewal or
proliferative capacities. A model of the stem cell compartment has
been proposed based on the functional capacities of the cell
(Hellman, S., et al., 1983, J. Clin. Oncol. 1:227-284).
Self-renewal would appear to be greater in those stem cells with
the shortest history of cell division, and this selfrenewal would
become progressively more limited with subsequent division of the
cells.
A human hematopoietic colony-forming cell with the ability to
generate progenitors for secondary colonies has been identified in
human umbilical cord blood (Nakahata, T. and Ogawa, M., 1982, J.
Clin. Invest. 70:1324-1328). In addition, hematopoietic stem cells
have been demonstrated in human umbilical cord blood, by colony
formation, to occur at a much higher level than that found in the
adult (Prindull, G., et al., 1978, Acta Paediatr. Scand.
67:413-416; Knudtzon, S., 1974, Blood 43(3):357-361). The presence
of circulating hematopoietic progenitor cells in human fetal blood
(Linch, D.C., et al., 1982, Blood 59(5):976-979) and in cord
blood
(Fauser, A. A. and Messner, H.A., 1978, Blood 52(6):1243-1248) has
also been shown. Human fetal and neonatal blood has been reported
to contain megakaryocyte and burst erythroblast progenitors
(Vainchenker, W., et al., 1979, Blood Cells 5:15-42), with
increased numbers of erythroid progenitors in human cord blood or
fetal liver relative to adult blood
(Hassan, M.W., et al., 1979, Br. J. Haematol. 41:477-484; Tchernia,
G., et al., 1981, J. Lab. Clin. Med. 97(3):322-331). Studies have
suggested some differences between cord blood and bone marrow cells
in the characteristics of CFU-GM (colony forming unit-granulocyte,
macrophage) which express surface Ia antigens (Koizumi, S., et al.,
1982, Blood 60(4):1046-1049).
U.S. Pat. No. 4,714,680 discloses cell suspensions comprising human
stem and progenitor cells and methods for isolating such
suspensions, and the use of the cell suspensions for hematopoietic
reconstitution.
2.2. Reconstitution of the Hematopoietic System
Reconstitution of the hematopoietic system has been accomplished by
bone marrow transplantation. Lorenz and coworkers showed that mice
could be protected against lethal irradiation by intravenous
infusion of bone marrow (Lorenz,
20 E., et al., 1951, J. Natl. Cancer Inst. 12:197-201). Later
research demonstrated that the protection resulted from
colonization of recipient bone marrow by the infused cells
(Lindsley, D.L., et al., 1955, Proc. Soc. Exp. Biol. Med.
90:512-515; Nowell, P.C., et al., 1956, Cancer Res. 16:258-261;
Mitchison, N.A., 1956, Br. J. Exp. Pathol. 37:239-247; Thomas,
E.D., et al., 1957, N. Engl. J. Med. 257:491-496). Thus, stem and
progenitor cells in donated bone marrow can multiply and replace
the blood cells responsible for protective immunity, tissue repair,
clotting, and other functions of the blood. In a successful bone
marrow transplantation, the blood, bone marrow, spleen, thymus and
other organs of immunity are repopulated with cells derived from
the donor.
U.S. Pat. No. 4,721,096 by Naughton et al. discloses a method of
hematopoietic reconstitution which comprises obtaining and
cryopreserving bone marrow, replicating the bone marrow cells in
vitro, and then infusing the cells into a patient. Bone marrow has
been used with increasing success to treat various fatal or
crippling diseases, including certain types of anemias such as
aplastic anemia (Thomas, E.D., et al., Feb. 5, 1972, The Lancet,
pp. 284-289), Fanconi,s anemia (Gluckman, E., et al., 1980, Brit.
J. Haematol. 45:557-564; Gluckman, E., et al., 1983, Brit. J.
Haematol. 54:431-440; Gluckman, E., et al., 1984, Seminars in
Hematology:21 (1):20-26), immune deficiencies (Good, R.A., et al.,
1985, Cellular Immunol. 82:36-54), cancers such as lymphomas or
leukemias (Cahn, J.Y., et al., 1986, Brit. J. Haematol. 63:457-470;
Blume, K.J. and Forman, S.J., 1982, J. Cell. Physiol. Supp.
1:99-102; Cheever, M.A., et al., 1982, N. Engl. J. Med.
307(8):479-481), carcinomas (Blijham, G., et al., 1981, Eur. J.
Cancer 17(4):433-441), various solid tumors (Ekert, H., et al.,
1982, Cancer 49:603-609; Spitzer, G., et al., 1980, Cancer
45:3075-3085), and genetic disorders of hematopoiesis. Bone marrow
transplantation has also recently been applied to the treatment of
inherited storage diseases (Hobbs, J.R., 1981, Lancet 2:735-739),
thalassemia major (Thomas, E.D., et al., 1982, Lancet 2:227-229),
sickle cell disease (Johnson, F.J., et al., 1984, N. Engl. J. Med.
311:780-783), and osteopetrosis (Coccia, P.F., et al., 1980, N.
Engl. J. Med. 302:701-708) (for general discussions, see Storb, R.
and Thomas, E. D., 1983, Immunol. Rev. 71:77-102; O'Reilly, R., et
al., 1984, Sem. Hematol. 21(3):188-221; 1969, Bone-Marrow
Conservation, Culture and Transplantation, Proceedings of a Panel,
Moscow, July 22-26, 1968, International Atomic Energy Agency,
Vienna; McGlave, P.B., et al., 1985, in Recent Advances in
Haematology, Hoffbrand, A.V., ed., Churchill Livingstone, London,
pp. 171-197).
Present use of bone marrow transplantation is severely restricted,
since it is extremely rare to have perfectly matched (genetically
identical) donors, except in cases where an identical twin is
available or where bone marrow cells of a patient in remission are
stored in a viable frozen state. Even in such an autologous system,
the danger due to undetectable contamination with malignant cells,
and the necessity of having a patient healthy enough to undergo
marrow procurement, present serious limitations. (For reviews of
autologous bone marrow transplantation, see Herzig, R.H., 1983, in
Bone Marrow Transplantation, Weiner, R.S., et al., eds., The
Committee On Technical Workshops, American Association of Blood
Banks, Arlington, Virginia; Dicke, K.A., et al., 1984, Sem.
Hematol. 21(2):109-122; Spitzer, G., et al., 1984, Cancer 54 (Sept.
15 Suppl.):1216-1225). Except in such autologous cases, there is an
inevitable genetic mismatch of some degree, which entails serious
and sometimes lethal complications. These complications are
two-fold. First, the patient is usually immunologically incapacited
by drugs beforehand, in order to avoid immune rejection of the
foreign bone marrow cells (host versus graft reaction). Second,
when and if the donated bone marrow cells become established, they
can attack the patient (graft versus host disease), who is
recognized as foreign. Even with closely matched family donors,
these complications of partial mismatching are the cause of
substantial mortality and morbidity directly due to bone marrow
transplantation from a genetically different individual.
Peripheral blood has also been investigated as a source of stem
cells for hematopoietic reconstitution (Nothdurtt, W., et al.,
1977, Scand. J. Haematol. 19:470-481; Sarpel, S.C., et al., 1979,
Exp. Hematol. 7:113-120; Ragharachar, A., et al., 1983, J. Cell.
Biochem. Suppl. 7A:78; Juttner, C.A., et al., 1985, Brit. J.
Haematol. 61:739-745; Abrams, R.A., et al., 1983, J. Cell. Biochem.
Suppl. 7A:53; Prummer, O., et al., 1985, Exp. Hematol. 13:891-898).
In some studies, promising results have been obtained for patients
with various leukemias (Reiffers, J., et al., 1986, Exp. Hematol.
14:312-315 (using cryopreserved cells); Goldman, J.M. et al., 1980,
Br. J. Haematol. 45:223-231; Tilly, H., et al., Jul. 19, 1986, The
Lancet, pp. 154-155; see also To, L.B. and Juttner, C.A., 1987,
Brit. J. Haematol. 66: 285-288, and references cited therein); and
with lymphoma (Korbling, M., et al., 1986, Blood 67:529-532). It
has been implied that the ability of autologous peripheral adult
blood to reconstitute the hematopoietic system, seen in some cancer
patients, is associated with the far greater numbers of circulating
progenitor cells in the peripheral blood produced after
cytoreduction due to intensive chemotherapy and/or irradiation (the
rebound phenomenon) (To, L.B. and Juttner, C.A., 1987, Annot.,
Brit. J. Haematol. 66:285-288; see also 1987, Brit. J. Haematol.
67:252-253, and references cited therein). Other studies using
peripheral blood have failed to effect reconstitution (Hershko, C.,
et al., 1979, The Lancet 1:945-947; Ochs, H.D., et al., 1981,
Pediatr. Res. 15(4 Part 2):601).
Studies have also investigated the use of fetal liver cell
transplantation (Cain, G.R., et al., 1986, Transplantation
41(1):32-25; Ochs, H.D., et al., 1981, Pediatr. Res. 15(4 part
2):601; Paige, C.J., et al., 1981, J. Exp. Med. 153:154-165;
Touraine, J.L., 1980, Excerpta Med. 514:277; Touraine, J.L., 1983,
Birth Defects 19:139; see also Good, R.A., et al., 1983, Cellular
Immunol. 82:44-45 and references cited therein) or neonatal spleen
cell transplantation (Yunis, E.J., et al., 1974, Proc. Natl. Acad.
Sci. U.S.A. 72:4100) as stem cell sources for hematopoietic
reconstitution. Cells of neonatal thymus have also been
transplanted in immune reconstitution experiments (Vickery, A.C.,
et al., 1983, J. Parasitol. 69(3):478-485; Hirokawa, K., et al.,
1982, Clin. Immunol. Immunopathol. 22:297-304).
2.3. Cryopreservation of Cells
Freezing is destructive to most living cells. Upon cooling, as the
external medium freezes, cells equilibrate by losing water, thus
increasing intracellular solute concentration. Below about
10-15.degree. C., intracellular freezing will occur. Both
intracellular freezing and solution effects are responsible for
cell injury (Mazur, P., 1970, Science 168:939-949). It has been
proposed that freezing destruction from extracellular ice is
essentially a plasma membrane injury resulting from osmotic
dehydration of the cell (Meryman, H.T., et al., 1977, Cryobiology
14:287-302).
Cryoprotective agents and optimal cooling rates can protect against
cell injury. Cryoprotection by solute addition is thought to occur
by two potential mechanisms: colligatively, by penetration into the
cell, reducing the amount of ice formed; or kinetically, by
decreasing the rate of water flow out of the cell in response to a
decreased vapor pressure of external ice (Meryman, H.T., et al.,
1977, Cryobiology 14:287-302). Different optimal cooling rates have
been described for different cells. Various groups have looked at
the effect of cooling velocity or cryopreservatives upon the
survival or transplantation efficiency of frozen bone marrow cells
or red blood cells (Lovelock, J.E. and Bishop, M.W.H., 1959, Nature
183:1394-1395; Ashwood-Smith, M.J., 1961, Nature 190:1204-1205;
Rowe, A.W. and Rinfret, A.P., 1962, Blood 20:636; Rowe, A.W. and
Fellig, J., 1962, Fed. Proc. 21:157; Rowe, A.W., 1966, Cryobiology
3(1):12-18; Lewis, J.P., et al., 1967, Transfusion 7(1):17-32;
Rapatz, G., et al., 1968, Cryobiology 5(1):18- 25; MaZur, p., 1970,
Science 168:939-949; Mazur, P., 1977, Cryobiology 14:251-272; Rowe,
A.W. and Lenny, L.L., 1983, Cryobiology 20:717; Stiff, P.J., et
al., 1983, Cryobiology 20:17-24; Gorin, N.C., 1986, Clinics in
Haematology 15(1):19-48).
The successful recovery of human bone marrow cells after long-term
storage in liquid nitrogen has been described (1983, American Type
Culture Collection, Quarterly Newsletter 3(4):1). In addition, stem
cells in bone marrow were shown capable of withstanding
cryopreservation and thawing without significant cell death, as
demonstrated by the ability to form equal numbers of mixed
myeloid-erythroid colonies in vitro both before and after freezing
(Fabian, I., et al., 1982, Exp. Hematol. 10(1):119-122). The
cryopreservation and thawing of human fetal liver cells (Zuckerman,
A.J., et al., 1968, J. Clin. Pathol. (London) 21(1):109-110), fetal
myocardial cells (Robinson, D.M. and Simpson, J.F., 1971, In Vitro
6(5):378), neonatal rat heart cells (Alink, G.M., et al., 1976,
Cryobiology 13:295-304), and fetal rat pancreases (Kemp, J.A., et
al., 1978, Transplantation 26(4):260-264) have also been
reported.
2.4. Gene Therapy
Gene therapy refers to the transfer and stable insertion of new
genetic information into cells for the therapeutic treatment of
diseases or disorders. The foreign gene is transferred into a cell
that proliferates to spread the new gene throughout the cell
population. Thus stem cells, or pluripotent progenitor cells, are
usually the target of gene transfer, since they are proliferative
cells that produce various progeny lineages which will potentially
express the foreign gene.
Most studies in gene therapy have focused on the use of
hematopoietic stem cells. High efficiency gene transfer systems for
hematopoietic progenitor cell transformation have been investigated
for use (Morrow, J.F., 1976, Ann. N.Y. Acad. Sci. 265:13; Salzar,
W., et al., 1981, in Organization and Expression of Globin Genes,
A.R. Liss, Inc., New York, p. 313; Bernstein, A., 1985, in Genetic
Engineering: Principles and Methods, Plenum Press, New York, p.
235; Dick, J.E., et al., 1986, Trends in Genetics 2:165). Reports
on the development of viral vector systems indicate a higher
efficiency of transformation than DNA-mediated gene transfer
procedures (e.g., CaPO.sub.4 precipitation and DEAE dextran) and
show the capability of integrating transferred genes stably in a
wide variety of cell types. Recombinant retrovirus vectors have
been widely used experimentally to transduce hematopoietic stem and
progenitor cells. Genes that have been successfully expressed in
mice after transfer by retrovirus vectors include human
hypoxanthine phosphoribosyl transferase (Miller, A., et al., 1984,
Science 255:630). Bacterial genes have also been transferred into
mammalian cells, in the form of bacterial drug resistance gene
transfers in experimental models. The transformation of
hematopoietic progenitor cells to drug resistance by eukaryotic
virus vectors, has been accomplished with recombinant
retrovirus-based vector systems (Hock, R.A. and Miller, A.D., 1986,
Nature 320:275-277; Joyner, A., et al., 1983, Nature 305:556-558;
Williams, D.A., et al., 1984, Nature 310:476-480; Dick, J.E., et
al., 1985, Cell 42:71-79); Keller, G., et al., 1985, Nature
318:149-154; Eglitis, M., et al., 1985, Science 230:1395-1398).
Recently, adeno-associated Virus vectors have been used
successfully to transduce mammalian cell lines to neomycin
resistance (Hermonat, P.L. and Muzyczka, N., 1984, supra;
Tratschin, J.-D., et al., 1985, Mol. Cell. Biol. 5:3251). Other
viral vector systems that have been investigated for use in gene
transfer include papovaviruses and vaccinia viruses (see Cline,
M.J., 1985, Pharmac. Ther. 29:69-92).
Other methods of gene transfer include microinjection,
electroporation, liposomes, chromosome transfer, and transfection
techniques (Cline, M.J., 1985, supra). Salser et al. used a
calcium-precipitation transfection technique to transfer a
methotrexate-resistant dihydrofolate reductase (DHFR) or the herpes
simplex virus thymidine kinase gene, and a human globin gene into
murine hematopoietic stem cells. In vivo expression of the DHFR and
thymidine kinase genes in stem cell progeny was demonstrated
(Salser, W., et al., 1981, in Organization and Expression of Globin
Genes, Alan R. Liss, Inc., New York, pp. 313-334).
Gene therapy has also been investigated in murine models with the
goal of enzyme replacement therapy. Thus, normal stem cells from a
donor mouse have been used to reconstitute the hematopoietic cell
system of mice lacking beta-glucuronidase (Yatziv, S., et al.,
1982, J. Lab. Clin. Med. 90:792-797). Since a native gene was being
supplied, no recombinant stem cells (or gene transfer techniques)
were necessary.
3. SUMMARY OF THE INVENTION
The present invention is directed to hematopoietic stem and
progenitor cells of neonatal or fetal blood, that are
cryopreserved, and the therapeutic uses of such stem and progenitor
cells upon thawing. In particular, the present invention relates to
the therapeutic use of fetal or neonatal stem cells for
hematopoietic (or immune) reconstitution. Hematopoietic
reconstitution with the cells of the invention can be valuable in
the treatment or prevention of various diseases and disorders such
as anemias, malignancies, autoimmune disorders, and other immune
dysfunctions and deficiencies. In another embodiment, fetal or
neonatal hematopoietic stem and progenitor cells which contain a
heterologous gene sequence can be used for hematopoietic
reconstitution in gene therapy.
In a preferred embodiment of the invention, neonatal or fetal blood
cells that have been cryopreserved and thawed can be used for
autologous (self) reconstitution.
The invention also relates to methods of collection and
cryopreservation of the neonatal and fetal stem and progenitor
cells of the invention.
3.1. DEFINITIONS
As used herein, the following abbreviations will have the meanings
indicated:
ACD=acid-citrate dextrose
BFU-E=burst-forming unit-erythroid. An hematopoietic progenitor
cell which is capable of producing a colony of erythroid progeny
cells in semi-solid medium.
BFU-E-1=an early erythroid progenitor cell, capable of producing a
colony of erythroid progeny cells in semi-solid medium upon
stimulation by erythropoietin, hemin (optional), and a
burst-promoting factor.
BFU-E-2=an erythroid progenitor cell, of greater maturity than
BFU-E-1, which is capable of producing a colony of erythroid
progeny cells in semi-solid medium upon stimulation by
erythropoietin and by hemin (optional).
CFU=colony-forming unit. A cell which is capable of producing a
colony of progeny cells in semi-solid medium.
CFU-GEMM=colony-forming unit-granulocyte, erythrocyte,
monocyte/macrophage, megakaryocyte. A multipotential hematopoietic
progenitor cell which is capable of producing a colony composed of
granulocyte, erythrocyte, monocyte/macrophage, and megakaryocyte
progeny, in semi-solid medium.
CFU-GM=colony-forming unit-granulocyte, macrophage. An
hematopoietic progenitor cell which is capable of producing a
colony composed of granulocyte and macrophage progeny in semi-solid
medium.
CFU-S=colony forming unit-spleen. A multipotential stem cell with
self-renewal capacity, which, upon inoculation into
lethally-irradiated mice, is capable of producing a colony (nodule)
on the spleen(s).
CPD=citrate-phosphate-dextrose
CSF=colony stimulating factor
DMSO=dimethyl sulfoxide
DNase=deoxyribonuclease
DPBS=phosphate buffered saline without magnesium or calcium
FCS=fetal calf serum
heterologous gene=a gene which is not present, or not functionally
expressed, in the designated host cell.
IMDM=Iscove's Modified Dulbecco's Medium
LD100/30 days=the minimum or near-minimal Lethal Dosage causing
100% mortality within a 30-day post-irradiation period
PHALCM=medium conditioned by phytohemagglutinin-stimulated
leukocytes from patients with hemochromatosis
PWMSCM=pokeweed mitogen spleen cell conditioned medium
S-cell=stem cell
SLE=systemic lupus erythematosus
.sup.3 HTdr=tritiated thymidine
TLI=total lymphoid irradiation
4. DESCRIPTION OF THE FIGURES
FIG. 1 presents data for neonatal blood volumes obtained in one
series of collections from individual births. The volume (ml) of
blood collected is shown along the X-axis, with infant weight (kg)
along the Y-axis. Open circles represent births by Caesarian
section; closed circles represent vaginal births.
FIG. 2 presents the data from neonatal blood volumes obtained in a
second series of collections from individual births. The volume
(ml) of blood collected is shown along the X-axis, with the infant
weight (kg) along the Y-axis. Closed circles represent vaginal
births, with collection by gravity drainage from the umbilical
cord. Open circles represent births by Caesarian section, with
collection by gravity drainage from the umbilical cord. Closed
triangles represent vaginal births, with collection from the
delivered placenta. Open triangles represent births by Caesarian
section, with collection from the delivered placenta.
FIGS. 3A and 3B are diagrammatic representations of the composition
of centrifuge tubes at different steps in a Ficoll-Hypaque density
separation, as described in Section 6.3.1, which can be employed to
obtain low density cells that are enriched in hematopoietic stem
and progenitor cells. The cord blood cell suspension is layered on
Ficoll-Hypaque before centrifugation (FIG. 3A). After
centrifugation, the low density cells appear as a sharp band
between the Ficoll-Hypaque and the phosphate-buffered saline (FIG.
3B).
FIG. 4 is a diagrammatic representation of the apparatus described
in Section 6.4, which can be used for the cryopreservation of
neonatal and fetal hematopoietic stem and progenitor cells. The
cryovials containing the cell suspensions are placed in a freezing
rack which is in turn placed in a 4.degree. C. methanol bath. The
methanol bath (in a metal or glass freezing dish) is in turn placed
in a -80.degree. C. freezer. After the cells reach the frozen
state, they are transferred to a long-term storage vessel
containing liquid nitrogen.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to hematopoietic stem and
progenitor cells of neonatal or fetal blood, that are
cryopreserved, and the therapeutic uses of such stem and progenitor
cells upon thawing.
In particular, the present invention relates to the use of fetal or
neonatal stem cells for hematopoietic reconstitution. In a
preferred embodiment of the invention, the fetal or neonatal stem
cells can be used in autologous hematopoietic reconstitution, i.e.,
in the reconstitution of the hematopoietic system of the same
individual from which they were originally derived. In such an
embodiment, the invention provides substantial advantages over the
present use of bone marrow for hematopoietic reconstitution.
Present use of bone marrow transplantation is severely restricted
by the fact that there is virtually never a perfectly matched
(genetically identical) donor, except in cases where an identical
twin is available or where bone marrow cells of, for example, a
cancer patient in remission are stored in the viable frozen state
in the hope that they will be free of malignant cells and healthy
enough to be returned to the patient for treatment of any future
relapse. Except in such cases, the inevitable genetic mismatch
which results can entail the serious and sometimes lethal
complications of host versus graft or graft versus host disease. In
order to avoid host rejection of the foreign bone marrow cells
(host versus graft reaction), the patient must be immunologically
incapacitated. Such immune incapacitation is itself a cause of
serious complications. Furthermore, when and if the donated bone
marrow cells become established, they can attack the patient (graft
versus host disease), who is recognized as foreign. Even with
closely matched family donors, these complications of partial
mismatching are the cause of substantial mortality and morbidity
directly due to bone marrow transplantation from a genetically
different individual.
In an embodiment of the invention directed to the use of neonatal
stem and progenitor cells for hematopoietic reconstitution, there
are several main reasons for preferring the use of such neonatal
cells to conventional bone marrow transplantation. First, no donor
is required because the cells can be obtained from neonatal blood
that would otherwise be discarded. Second, in a preferred
autologous system, i.e., involving use of "self" neonatal cells,
the complications arising in conventional bone marrow
transplantation from the need for pretransplantation drug-induced
or irradiation-induced immune incapacitation and from acute and
chronic graft-versus-host disease are all eliminated because, in
this embodiment, neonatal cells are returned to their original
owner and are therefore totally compatible. For these reasons,
present restrictions on the use of bone marrow transplantation
arising from difficulties in finding even approximately matched
donors, and from disease and mortality due to unavoidable genetic
incompatibility, do not apply to self-reconstitution with neonatal
cells. Third, regarding the preferred autologous embodiment, the
efficiency of genetically identical (self) cells in bone marrow
transplantation in animals is numerically many times greater than
that of cells from a genetically dissimilar donor (Balner, H.,
1977, Bone Marrow Transplantation and Other Treatment after
Radiation Injury, Martinus Nijhoff Medical Division, The Hague),
thus far fewer self cells are required for successful
reconstitution in the preferred autologous system.
Furthermore, the prospects of success in bone marrow
transplantation decline with age; although it is not clear whether
the age of donor or patient is more important, it is proper to
infer that younger (neonatal) cells are preferable for
hematopoietic reconstitution. Such neonatal or fetal cells have not
been subjected to the "environmental outrage" that adult cells have
undergone. Also, as an example of novel medical applications which
may be feasible with neonatal cells but not with conventional bone
marrow transplantation, restoration with self cells taken at birth
can be valuable in the treatment of disorders such as declining
immune responsiveness and autoimmunity (immune reactions against
one's own tissues) which occur in increasing frequency with
age.
Many of the relative disadvantages discussed supra of the use of
bone marrow cells for hematopoietic reconstitution, also apply to
the use of adult peripheral blood for such reconstitution, and
thus, the use of neonatal cells for hematopoietic reconstitution
according to the present invention provides distinct advantages
over the employment of adult peripheral blood. It has been implied
that the ability of autologous peripheral adult blood to
reconstitute the hematopoietic system, seen in some cancer
patients, is associated with the far greater numbers of circulating
progenitor cells in the peripheral blood produced after
cytoreduction due to intensive chemotherapy and/or irradiation (the
rebound phenomenon) (To, L.B. and Juttner, C.A., 1987, Annot.,
Brit. J., Haematol. 66:285-288; see also 1987, Brit. J. Haematol.
67:252-253, and references cited therein). There are possible
detrimental effects, known or unknown, of prior chemotherapy or
irradiation, on the stem and progenitor cell populations found in
these patients.
There are additional reasons for preferring the use of neonatal
cells for hematopoietic reconstitution as provided by the present
invention. Neonatal blood is a preferred source of cells for
hematopoietic reconstitution, since it is free from viral and
microbial agents, known or unknown, latent or otherwise, that may
be encountered in later life, other than those transmitted from the
mother or during labor and delivery. In addition, in view of the
extent to which the hematopoietic stem cell may possibly share with
other cells the limitation in total number of cell divisions that
it may undergo before senescence, it is proper to assume that the
neonatal hematopoietic stem cell has a self-renewal and
reconstituting capacity that is at least as great, and perhaps
greater, than that of hematopoietic stem cells obtained at any
later time in life.
In adults, stem and progenitor cells are mostly confined to the
bone marrow; very few circulate in the blood. In the newborn human
or animal, however, stem and progenitor cells circulate in the
blood in numbers similar to those found in adult bone marrow.
Doubtless this reflects the great demands for blood formation of
the growing infant. We calculate that the restorative capacity of
neonatal blood contained in the human umbilical cord and placenta,
which are customarily discarded at birth, equals or exceeds that of
the average donation of an adult's bone marrow. The efficacy of
human neonatal blood cells compared with adult bone marrow cells is
gauged by laboratory assays for stem cells and progenitor cells.
Progenitor cell assays imply that the reconstituting potential of
cells from 50 ml of cord blood (readily obtainable) is at least
equivalent to the average number of progenitor cells from adult
bone marrow that is used in autologous hematopoietic reconstitution
(see Section 6.8, infra). `S-cells`, representing probably the
earliest developmental form of the stem cell, are demonstrable in
human (cord) blood (Nakahata, T. and Ogawa, M., 1982, J. Clin.
Invest. 70:1324-1328). Thus, the cells of neonatal blood can be
judged an effective clinical substitute for adult bone marrow.
In laboratory animals, the efficacy of neonatal cells can be tested
directly. Accordingly we have shown that circulating neonatal
cells, in numbers lower than are contained in the cord and
placenta, will completely and permanently repopulate the entire
blood-forming and immune systems of a lethally irradiated adult
animal, promoting complete recovery and return to normal health
(see Section 6.11, infra).
The method of the invention may be divided into the following
stages solely for the purpose of description: (a) isolation of
fetal or neonatal hematopoietic stem and progenitor cells; (b)
inspection and testing of fetal or neonatal blood; (c) enrichment
for hematopoietic stem and progenitor cells; (d) cryopreservation;
(e) recovery of stem and progenitor cells from the frozen state;
(f) examination of cells recovered for clinical therapy; and (g)
therapeutic uses in reconstitution of the hematopoietic system.
Since both fetal and neonatal hematopoietic cells are envisioned
for use in the present invention, descriptions and embodiments of
the invention herein described for neonatal cells are meant to
apply equally to fetal cells, unless clearly otherwise indicated or
apparent.
5.1. Isolation of Fetal or Neonatal Hematopoietic Stem and
Progenitor Cells
Fetal or neonatal blood are sources of the hematopoietic stem and
progenitor cells of the present invention.
Fetal blood can be obtained by any method known in the art. For
example, fetal blood can be taken from the fetal circulation at the
placental root with the use of a needle guided by ultrasound
(Daffos, F., et al., 1985, Am. J. Obstet Gynecol 153:655-660;
Daffos, F., et al., 1983, Am. J. Obstet. Gynecol. 146:985), by
placentocentesis (Valenti, C., 1973, Am. J. Obstet. Gynecol.
115:851; Cao, A., et al., 1982, J. Med. Genet. 19:81), by fetoscopy
(Rodeck, C.H., 1984, in Prenatal Diagnosis, Rodeck, C.H. and
Nicolaides, K.H., eds., Royal College of Obstetricians and
Gynaecologists, London), etc.
In a preferred embodiment of the invention, neonatal hematopoietic
stem and progenitor cells can be obtained from umbilical cord blood
and/or placental blood. The use of cord or placental blood as a
source of cells to repopulate the hematopoietic system provides
numerous advantages. Cord blood can be obtained easily and without
trauma to the donor. In contrast, at present, the collection of
bone marrow cells for transplantation is a traumatic experience
which is costly in terms of time and money spent for
hospitalization. Cord blood cells can be used for autologous
transplantation, when and if needed, and the usual hematological
and immunological problems associated with the use of allogeneic
cells, matched only partially at the major histocompatibility
complex or matched fully at the major, but only partially at the
minor complexes, are alleviated.
Collections should be made under sterile conditions. Immediately
upon collection, the neonatal or fetal blood should be mixed with
an anticoagulent. Such an anticoagulent can be any known in the
art, including but not limited to CPD (citrate-phosphate-dextrose),
ACD (acid citrate-dextrose), Alsever's solution (Alsever, J.B. and
Ainslie, R.B., 1941, N. Y. St. J. Med. 41:126), De Gowin's Solution
(De Gowin, E.L., et al., 1940, J. Am. Med. Ass. 114:850),
Edglugate-Mg (Smith, W.W., et al., 1959, J. Thorac. Cardiovasc.
Surg. 38:573), Rous-Turner Solution (Rous, P. and Turner, J.R.,
1916, J. Exp. Med. 23:219), other glucose mixtures, heparin, ethyl
biscoumacetate, etc. (See Hurn, B.A.L., 1968, Storage of Blood,
Academic Press, New York, pp. 26-160). In a preferred embodiment,
ACD can be used.
5.1.1. Collection of Neonatal Blood
The object of this aspect of the invention is to obtain a neonatal
blood collection of adequate volume that is free of contamination.
Since umbilical cord blood is a rich source of stem and progenitor
cells (see Section 6.6, infra; Nakahata, T. and Ogawa, M., 1982, J.
Clin. Invest. 70:1324-1328; Prindull, G., et al., 1978, Acta.
Paediatr. Scand. 67:413-416; Tchernia, G., et al., 1981, J. Lab.
Clin. Med. 97(3):322-331), the preferred source for neonatal blood
is the umbilical cord and placenta. The neonatal blood can
preferably be obtained by direct drainage from the cord and/or by
needle aspiration from the delivered placenta at the root and at
distended veins.
5.1.1.1. Volume
In a preferred embodiment, volumes of 50 ml or more of neonatal
blood are obtained (see Section 6.1, infra).
Practical experience indicates that volumes of 50 ml or more are
easily collected without additional measures in 80% of term births,
and that collections of more than 40 ml are obtainable more than
90% of the time. Lower volumes may also be acceptable, and
indicated under some circumstances (see Sections 5.1.1.2.3.1 and
5.1.1.2.3.2, infra).
The following information suggests that as little as 50 ml of cord
blood contains enough of the appropriate cells to repopulate the
hematopoietic system of an adult, and it is possible that even less
cord blood would have the same effect:
1. In a small sampling of cases for autologous marrow
transplantation (Spitzer, G., et al., 1980, Blood 5:317-323), rapid
repopulation of hematopoiesis in patients with acute leukemia was
associated with as few as 0.24 million granulocyte-macrophage
progenitor cells (CFU-GM).
2. In human cord blood, there are approximately 50-200 CFU-GM per
100,000 low density cells and at least 5 million low density cord
blood cells per milliliter. Thus 50 milliliters of cord blood would
contain in the range of 0.1 to greater than 0.5 million CFU-GM (see
also Section 6.8, infra). The upper value agrees closely with
estimations from the number of CFU-GM in 12.5 to 19 day old fetal
blood (Lynch, D.C., et al., 1982, Blood 59:976-979).
3. Importantly, stem and progenitor cells in cord blood appear to
have a greater proliferative capacity in culture dishes than those
in adult bone marrow (Salahuddin, S.Z., et al., 1981, Blood
58:931-938; Cappellini, M.D., et al., 1984, Brit. J. Haematol.
57:61-70).
Significant to the use of cord blood as a source of stem cells, is
that the assay for S-cells has been adapted for the growth of human
cord blood (Nakahata, T. and Ogawa, M., 1982, J. Clin. Invest.
70:324-1328). All the known progenitor cells are present in cord
blood in high numbers and this includes those progenitors for
multilineages, granulocytes, macrophages, erythrocytes, mast cells,
and basophils (id.; Fauser, A.A. and Messner, H.A., 1978, Blood
52:1243-1248; Koizumi, S., et al., 1982, Blood 60:1046-1049;
Prindull, G., et al., 1978, Acta Paediatr. Scand. 67:413-416).
Furthermore, hematopoietic stem and progenitor cells can
potentially be multiplied in culture, before or after
cryopreservation, (see Sections 5.1.3.2, 5.3.2, infra), thus
expanding the number of stem cells available for therapy.
5.1.1.2. Preferred Aspects
The following subsections provide detailed descriptions of
preferred particular embodiments of the invention, and are intended
for descriptive purposes only, in no way limiting the scope of the
invention.
5.1.1.2.1. Collection Kit
In a preferred aspect, a collection kit, packaged in a sterile
container, can be used. In one particular embodiment, the
collection kit can consist of:
(i) a wide-mouth, graduated, collection container, with
anticoagulant, into which the cut end of the cord may be placed for
collection by gravity drainage. A small funnel can be provided for
use if needed.
(ii) (optional) a plastic, flexible, sealed collection bag, similar
to a donation bag, which has ports for injection of the collected
blood, and contains anticoagulant.
(iii) an identification label, which identifies the infant source
of the sample and time of collection.
For multiple births, separate collections, each performed with a
separate kit, are preferred.
Sterilization of the containers can occur by any technique known in
the art, including but not limited to beta-irradiation, autoclaving
of suitable materials in a steam sterilizer, etc. For example, in a
preferred embodiment, sterilization by beta-irradiation can be
carried out by exposure to 2.5 megarads from a tungsten source (see
Section 6.1, infra).
The collection kit may be placed in the surgical field in advance
of a delivery, to afford ready availability.
5.1.1.2.2. Vaginal Delivery of the Term Infant
Vaginal delivery of the normal infant at term, spontaneously, by
forceps, or as a breech delivery, should allow an ample collection
of cord blood. After clamping the cord, the volume of fetal blood
remaining in the cord and attached placenta has been estimated at
45 ml/kg infant body weight, or approximately 145 ml for a 7 lb
(3.2 kg) baby (Hellman, L.M., et al., 1971, Williams Obstetrics,
14th Ed., Appleton-Century-Crofts, New York, p. 216).
Following delivery of the infant, by any method, with or without
anesthesia, the infant is held in the plane of the vagina, and the
cord is doubly cross-clamped and cut approximately three inches
(7-8 cm) from the umbilicus. The infant is removed.
Maintaining usual sterile precautions, the cord is then transected
just above the crushed portion in the clamp, and the resulting flow
of fetal blood from umbilical vessels is caught in the container
provided. An adequate collection can usually be accomplished
without milking the cord, and is complete in approximately two
minutes, before placental separation has occurred. Care should be
taken to avoid contamination by maternal blood, urine, or other
fluids in the delivery field. Blood in the container is then
transferred to the bag provided for transport to the storage
facility or, alternatively, the original container, if equipped
with a tight screw cap, can itself be sent to the storage facility
without transfer of its contents.
If, following infant delivery, events make collection at that time
undesirable, collection can be done after delivery of the placenta
(see Section 5.1.1.2.3.6, infra). If maternal infection is
suspected, such a placental collection may be preferable.
Collection can also be carried out by aspiration from the delivered
placenta, in addition to gravity drainage.
In a most preferred embodiment, immediate cord clamping after
delivery is carried out, in order to achieve collection of the
greatest possible volume of cord blood. Studies have shown that the
relative distribution of blood between the infant and placental
circuits gradually shifts to the infant's blood circuits with
increasing delay in cord clamping after delivery (Yao, A.C., et
al., Oct. 25, 1969, Lancet 871-873).
5.1.1.2.3. Other Circujmstances of Birth and Delivery
5.1.1.2.3.1. Premature Birth
The cord blood of premature infants may contain an even greater
proportion of stem and progenitor cells than full-term cord blood.
Consequently, smaller volumes of cord blood from premature infant
delivery may give as good a yield of stem and progenitor cells.
(The use of stem and progenitor cell assays as described in
Sections 5.4.2 and 6.6 can determine the yield). Thus, in general,
cord blood collection should be carried out if premature infant
survival is anticipated, even though the volume of blood collected
may be less than usual. Collection procedures should be the same as
for term births.
5.1.1.2.3.2. Multiple Births
Cord blood collections undertaken at the time of multiple births
involve additional procedural considerations:
(i) Multiple births are often premature, and volumes of cord blood
will be correspondingly smaller. Collections should be made
nevertheless, so that the decision to preserve for storage can be
made later.
(ii) When births of two or more infants occur, where use of the
cord collection is envisioned for later self-reconstitution, it is
essential that each cord collection be identified with the proper
infant. In cases of doubtful zygosity, blood typing can be done on
cord blood and postnatal samples.
(iii) The timing of twin cord blood collection can be at the
discretion of the obstetrician (after delivery of one twin; or
after delivery of both).
(iv) A careful description of the placental relationships should be
made (single or double amnions; single, double or fused
chorions).
5.1.1.2.3.3. Caesarian Delivery
Cord blood collections at caesarean section can be carried out with
the same kit, and with the same procedure as vaginal delivery. The
cut end of the cord is lowered to promote gravity drainage.
At caesarean section, it is strongly preferred that the cord blood
collection be made after delivery of the infant, and before
placental separation. However, this may not be desirable in some
instances, such as where there is brisk hemorrhage, the need to
incise or separate an anteriorly implanted placenta, or
preoccupation of personnel with other events in the operating
field. Thus, in these and similar cases, the placenta can be
removed, and cord blood collected from it later.
5.1.1.2.3.4. Complicated Delivery
Complications of delivery arising from the condition of the mother
or the infant, or both, may require the immediate and urgent
attention of the obstetrician and his assistants. Under these
circumstances, the delivered placenta can be placed to one side,
and collection carried out as soon as feasible.
5.1.1.2.3.5. Abnormal Placenta
For successful cord blood collection, it is preferred that the
placenta be intact, or nearly so. Cases of marginal or partial
separation can still offer an opportunity for collection, although
it may have to be carried out after delivery of the placenta, if
clinical circumstances indicate a need for prompt removal.
Collections will be disfavored for use if a rupture of fetal
circulation has occurred. Samples can be tested later for
contamination by maternal blood (see Section 5.1.2, infra).
Accurate description of the placental abnormality is preferred.
5.1.1.2.3.6. Collection From the Delivered Placenta
When rapid delivery of the placenta occurs or becomes necessary,
and cord blood collection cannot be accomplished prior to placental
separation, a sample of sufficient volume can still be obtained
after delivery. The placenta and attached cord, still clamped, are
placed to one side, but still within the sterile field. Collection
is by the same technique described supra in section 5.1.1.2.2. It
is preferred, however, that collection be completed within five
minutes of delivery, while maintaining sterile procedures.
Cord blood collection prior to placental separation is preferred
over collection from the delivered placenta for the following
reasons: In a collection from delivered placenta, (i) collection
volumes are generally less; (ii) some degree of clotting in the
placental circulation may restrict recovery, and (iii) the
likelihood of contamination, by maternal blood or other agents, is
increased. Therefore, the determination of suitability of the
sample collected from a delivered placenta is especially
important.
5.1.1.2.3.7. Meical Conditions of the Mother
Given the general prohibition against maternal use of drugs which
would adversely affect the fetus, it is unlikely that maternal
therapy or medical status in the general sense would adversely
affect stem cell retrieval from cord blood collection of a normal
infant. In a preferred embodiment, however, specific information
should be obtained in regard to drug abuse, viral diseases capable
of vertical transmission, and the influence of acute maternal
illness at the time of delivery, since it is possible that these
may affect stem cell retrieval from cord blood.
5.1.1.2.3.8. Unplanned Delivery
Despite elaborate plans, delivery may occur inopportunely,
sometimes prematurely, and without the immediate services of a
physician. Under these circumstances, the following procedures are
preferred: (i) cord blood collection should be attempted with the
standard kit, described supra; (ii) the placenta, if delivered on
an unsterile field, should simply be kept as clean as possible,
left with the cord clamped, and collection attempted within 5
minutes; (iii) the cord should be wiped with a cleansing agent
(e.g. Betadine), and transected above the clamp, to make the
collection; and (iv) circumstances of the delivery should be
described with the specimen.
5.1.1.2.4. Recordation of Data
In a preferred embodiment, the data listed in Table I, infra, are
obtained at the time of collection in order to ensure the accurate
identification and evaluation of the collected blood.
TABLE I ______________________________________ DATA TO BE RECORDED
AT THE TIME OF NEONATAL BLOOD COLLECTION
______________________________________ Date and time of delivery
Full name and address of mother Hospital identification Sex of
infant Weight of infant Birth order (for multiple pregnancies)
Gestational age Pregnancy complications Intrapartum complications
Type of delivery Placental collection (amount of blood collected)
Placental description and weight Condition of infant
______________________________________
5.1.2. Inspection and Testing of Neonatal Blood
In a preferred embodiment, the neonatal blood sample is inspected
and tested to ensure its suitability. Appropriate inspections and
tests include but are not limited to the procedures described
infra.
If the blood collection sample is to be shipped to a processing
plant, the blood container and its contents should be inspected for
defects such as inadequate closure and leakage. As an option, the
collection kit may include a suitably positioned reusable
maximum-minimum mercury thermometer to register the range of
temperature change during shipment. Clots, opacity of the plasma
and visible hemolysis are indications of bacterial contamination or
other consequences of faulty handling. Time elapsed since
collection can be noted.
The following tests on the collected neonatal blood sample can be
performed either routinely, or where clinically indicated:
(i) Bacterial culture: To ensure the absence of microbial
contamination, established assays can be performed, such as routine
hospital cultures for bacteria under aerobic and anaerobic
conditions.
(ii) Diagnostic screening for pathogenic microorganisms: To ensure
the absence of specific pathogenic microorganisms, various
diagnostic tests can be employed. Diagnostic screening for any of
the numerous pathogens transmissible through blood can be done by
standard procedures. As one example, the collected blood sample can
be subjected to diagnostic screening for the presence of Human
Immunodeficiency Virus (HIV), the causative agent of Acquired
Immune Deficiency Syndrome (AIDS) (Gallo et al., 1984, Science
224:500-503; Barre-Sinoussi, F., et al., 1983, Science 220:868;
Levy, J.A., et al., 1984, Science 225:840). Any of numerous assay
systems can be used, based on the detection of virions,
viral-encoded proteins, HIV-specific nucleic acids, antibodies to
HIV proteins, etc.
(iii) Confirmation of neonatal origin of the blood: Contamination
with maternal blood, not necessarily a contraindication to storage
and clinical utility, may be suspected from the obstetrical
history. Presence of maternal cells, and of adult blood generally,
can be revealed by various tests, including but not limited to I
typing (Wiener, A.S., et al., 1965, Am. J. Phys. Anthropol. 23(4):
389-396); analysis on a Coulter Channelyzer, which detects size
differences between neonatal and maternal blood cells (Daffos, F.,
et al., 1985, Am. J. Obstet. Gynecol. 153:655-660); staining
procedures for hemoglobin such as the Kleinhauer-Betke technique
(Betke, K., 1968, Bibl. Haematologica 29:1085) and others (Clayton,
E.M., et al., 1970, Obstetrics and Gynecology 35(4):642-645), which
detect differences in the types of hemoglobin contained in red
blood cells before birth versus in later life; etc.
In a preferred embodiment, I typing can be done by established
methods, such as agglutination with anti-i and anti-I antibodies.
Erythrocytes of neonates are i strong, I weak; by 18 months of age,
erythrocytes are I strong; i weak (Marsh, W.L., 1961, Brit. J.
Haemat. 7:200). Thus, the degree of reaction with anti-i or anti-I
antibodies is a measure of the proportion of neonatal blood and red
cells in a mixture of neonatal and adult blood. The corresponding
contamination with maternal stem and progenitor cells would be far
less than the total maternal cell contamination since the stem and
progenitor cells are rare in adult blood. (Scarcity of stem and
progenitor cells in colony assays (see Sections 5.4.2 and 6.6,
infra) is another distinction between neonatal and adult
blood.)
5.1.3. Optional Procedures
In a preferred embodiment of the invention, whole neonatal blood,
as collected, can be cryogenically frozen, thus minimizing cell
losses which can be incurred during cell processing protocols.
However, cell separation procedures and expansion of stem and
progenitor cells in in vitro cultures remain options. Such
procedures may be useful, e.g., in reducing the volume of sample to
be frozen, and increasing cell count, respectively. The procedures
described infra in Sections 5.1.3.1 and 5.1.3.2 should be carefully
screened before use, in order to ensure that hematopoietic stem and
progenitor cell loss in processing does not endanger the
therapeutic efficacy of a collected blood sample in hematopoietic
reconstitution.
5.1.3.1. Enrichment for Hematopoietic Stem and Progenitor Cells:
Cell Separation Procedures
After receiving cord blood or bone marrow samples in anticoagulant
(=.g., ACD), the cells can be subjected to physical and/or
immunological cell separation procedures. Such procedures enrich
for the hematopoietic stem and progenitor cells so that fewer total
cells have to be stored and transplanted. However, if cell
separation is desired, care should be taken to ensure sufficient
recovery of the hematopoietic stem and progenitor cells.
Various procedures are known in the art and can be used to enrich
for the stem and progenitor cells of the present invention. These
include but are not limited to equilibrium density centrifugation,
velocity sedimentation at unit gravity, immune rosetting and immune
adherence, counterflow centrifugal elutriation, T lymphocyte
depletion, and fluorescence-activated cell sorting, alone or in
combination. Recently, procedures have been reported for the
isolation of very highly enriched populations of stem/progenitor
cells. Murine CFU-S have been purified by several groups using
slightly different procedures (Visser, J.W.M., et al., 1984, J.
Exp. Med. 59:1576; Nijhof, W., et al., 1984, Exp. Cell Res.
155:583; Bauman, J.G.J., et al., 1986, J. Cell. Physiol. 128:133;
Lord, B.I. and Spooncer, E., 1986, Lymphokine Res. 5:59). Studies
using human (Emerson, S.G., et al., 1985, J. Clin. Invest. 76:1286)
or murine (Nicola, N.A., et al., 1981, Blood 58:376) fetal liver
cells have yielded highly enriched progenitor cells with up to 90%
of them being colony forming cells for multi-, erythroid-, and
granulocyte-macrophage lineages. CFU-E have also been very highly
enriched (Nijhof, W., et al., 1983, J. Cell Biol. 96:386).
Purification of adult mouse marrow CFU-GM with cloning efficiencies
of up to 99% in semi-solid medium has been accomplished by
pretreatment of mice three days prior to sacrifice with
cyclophosphamide, density separation of cells on Ficoll-Hypaque,
and counterflow centrifugal elutriation (Williams, D.E., et al.,
1987, Exp. Hematol. 15:243). The resulting fraction of cells
contained no detectable CFU-GEMM, BFU-E or CFU-MK, but up to 10% of
the cells formed CFU-S measured at day 12. These procedures, or
modifications thereof, can be used, and are within the scope of the
present invention.
Human stem and progenitor cells are present in the non-adherent,
low density, T-lymphocyte-depleted fraction of bone marrow, spleen,
and (adult and cord) blood cells. In a specific embodiment, low
density (density less than 1.077 gm/cm.sup.3) cells can be
separated by use of Ficoll-Hypaque (Pharmacia Fine Chemicals,
Piscataway, NJ) (see Section 6.3.1, infra) or Percol (Broxmeyer,
H.E., 1982, J. Clin. Invest. 69:632-642). In this procedure, the
mature cells of the granulocytic series, which are not needed for
transplantation, are removed in the dense fraction which goes to
the bottom of the tube. An adherence/nonadherence separation
protocol can also be used for enrichment of hematopoietic stem and
progenitors; protocols which can be used are described in Section
6.3.2, infra, and in Broxmeyer, H.E., et al., 1984, J. Clin.
Invest. 73:939-953, which is incorporated by reference herein.
If desired, autologous plasma can be removed for use in the
freezing process. In particular, the blood or marrow samples can be
allowed to settle at unit gravity in a test tube. The setting
process can be hastened by addition of sterile-pyrogen-free Dextran
Sulphate. After approximately 15 minutes, the upper layer
containing the nucleated cells in plasma can be removed and
centrifuged (e.g., 200-400 X g). The nucleated cells pellet to the
bottom of the tube and the plasma is removed and stored in a tube
at 4.degree. C. The nucleated cells are washed, counted and, if
desired, further separated (e.g., by use of a density "cut"
procedure with Ficoll-Hypaque or Percol).
In order to enrich hematopoietic stem and progenitor cells, it is
also possible to use cell separation procedures that entail
immunological recognition of cells. Stem and progenitor cells can
be isolated by positive or negative selection using antibodies that
recognize antigenic determinants on the surface of cells. One means
is to separate the cells by using monoclonal antibodies which
recognize cell surface determinants on these cells, in conjunction
with separation procedures such as fluorescence-activated cell
sorting or panning (Broxmeyer, H.E., et al., 1984, J. Clin. Invest.
73:939-953). At present, there are no known antigenic determinants
that are absolutely specific for human hematopoietic stem and
progenitor cells. However, these cells do contain antigenic
determinants that are not present on all other cells, which can be
used in antibody selection protocols for enrichment purposes; such
antigens include but are not limited to those described infra.
Within the human system, several antigens have been found on
stem/progenitor cells. The first antigenic system studied
intensively was that of the MHC class II antigens, especially
HLA-DR. This has been found on CFU-GEMM, BFU-E, and CFU-GM (Lu, L.,
et al., 1983, Blood 61:250; Winchester, R.J., et al., 1977, Proc.
Natl. Acad. Sci. U.S.A. 74:4012; Busch, F.W., et al., 1987, Blut
54:179). Several investigators have suggested that HLA-DR are not
found, or are present at a low density on cells earlier than
CFU-GEMM (Moore, M.A.S., et al., 1980, Blood 55:682; Keating, A.,
et al., 1984, Blood 64:1159) but others have not agreed (e.g.,
Falkenberg, J.H.F., et al., 1985, J. Exp. Med. 162:1359). This
discrepancy may be due to the existence of specific subsets of
early progenitors. In fact, the expression of HLA-DR is higher
during the S-phase of the cell cycle of hematopoietic progenitor
cells (Broxmeyer, H.E., 1982, J. Clin. Invest. 69:632; Cannistra,
S.A., et al., 1985, Blood 65:414). Day 14 CFU-GM express higher
levels of HLA-DR than day 7 CFU-GM, and among day 7 CFU-GM,
monocyte progenitors express more HLA-DR than do the granulocyte
progenitors (Griffin, J.D., et al., 1985, Blood 66:788). Expression
of HLA-DR decreases and is lost during early myeloid precursor cell
states and it has been suggested that HLA-DR antigens might play a
role in myeloid development (Winchester, R.J., et al., 1977,
supra).
Groups of antibodies have been used to distinguish different
progenitors of the granulocyte-macrophage lineage (Ferrero, D., et
al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4114). Type 1 CFU-GM
contribute all of the peripheral blood CFU-GM, as well as a small
number of bone marrow CFU-GM. They express surface antigens
recognized by S3-13 and S17-25 antibodies, but not by RIB19 and
WGHS-29-1 antibodies. Type 2 CFU-GM are present only in the marrow
and react with S3-13, RIB19, and WGHS-29-1. Culture of type 1
CFU-GM in liquid culture generates type 2 CFU-GM. These antibodies
have also been used to characterize CFU-GM from patients with
chronic myeloproliferative disorders (Robak, T., et al., 1985,
Leukemia Res. 9:1023; Ferrero, D., et al., 1986, Cancer Res.
46:975).
Other antigens on human stem/progenitor cells include those
reactive with the My10 (Leary, A.G., et al., 1987, Blood 69:953;
Strauss, L.C., et al., 1986, Exp. Hematol. 14:879), 3C5 (Katz,
F.E., et al., 1985, Leukemia Res. 9:191; Katz, F.E., et al., 1986,
Leukemia Res. 10:961), RFB-1 (Bodger, M.P., et al., 1983, Blood
61:1006), 12-8 (Andrews, R.G., et al., 1986, Blood 67:842), and
L4F3 (Andrews, R.G., et al., 1986, Blood 68:1030) antibodies. The
antigen recognized by L4F3 is on CFU-GM, CFU-MK, BFU-E, and
CFU-GEMM, but is apparently absent from cells which generate these
progenitors in suspension culture (id.). L4F3 reacts with most
blast cells from patients with acute myelogenous leukemia, and
treatment of cells from such patients with L4F3 has allowed the
growth of normal progenitor cells in vitro (Bernstein, I.D., et
al., 1987, J. Clin. Invest. 79:1153). The antigen recognized by
another antibody, Myll, is expressed on CFU-GM, but not on BFU-E or
CFU-GEMM (Strauss, L.C., et al., 1986, Exp. Hematol. 14:935).
Receptors for various lectins are also expressed on
stem/progenitors (Nicola, N.A., et al., 1980, J. Cell Physiol.
103:217; Reisner, Y., et al., 1982, Blood 59:360; Reisner, Y., et
al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:2933; Aizawa, S., and
Tavassoli, M., 1986, Int. J. Cell Cloning 4:464).
Some success in enriching adult human bone marrow progenitor cells
has been reported based on the use of monoclonal antibodies and
cell sorting. In some studies, cells have been sorted only on
positive versus negative populations (Katz, F.E., et al., 1986,
Leukemia Res. 10:961). Recently, My10 and HLA-DR antibodies were
used in association with two color sorting to obtain highly
enriched progenitor cell populations from human marrow (Lu, L., et
al., 1987, J. Immunol.139(6):1823-1829).
In specific embodiments, antibodies which are currently available
and can be used in enrichment protocols include My-10, 3C5, or
RFB-1. These antibodies can be used alone or in combination with
procedures such as "panning" (Broxmeyer, H.E. et al., 1983, J.
Clin. Invest. 73:939-953) or fluorescence activated cell-sorting
(FACS) (Williams, D.E., et al., 1985, J. Immunol. 135:1004; Lu, L.,
et al., 1986, Blood 68(1):126-133) to isolate those cells
containing surface determinants recognized by the monoclonal
antibodies.
In another embodiment, enrichment, if desired, can proceed by the
use of monoclonal antibodies to major histocompatibility (MHC)
class II antigens (especially HLA-DR) and to My10 (Lu, L., et al.,
1987, J. Immunol. 139(6): 1823-1829.
T lymphocyte depletion can also be used to enrich for hematopoietic
stem or progenitor cells. In this procedure, T lymphocytes are
selectively removed from the cell population by pretreating cells
with a monoclonal antibody(ies), that recognize a T cell antigen,
plus complement. Such a procedure has been described previously
(Broxmeyer, H.E., et al., 1984, J. Clin. Invest. 73:939-953).
Another method that can be used is that of separating the stem and
progenitor cells by means of selective agglutination using a lectin
such as soybean (Reisner, Y., et al., 1980, Proc. Natl. Acad. Sci.
U.S.A. 77:1164). This procedure can be a viable alternative for
separation and enrichment of stem and progenitor cells without
removal of possibly necessary accessory cells (Reisner, Y., et al.,
1983, Blood 61(2):341-348; Reisner, Y., et al., 1982, Blood
59(2)::360-363).
Theoretically, only one early stem cell is needed for repopulation
of the entire hematopoietic system. There is laboratory evidence
that under ideal conditions and when the microenvironment nurturing
the stem and progenitor cells in the recipient animal is not
affected, a single stem cell can entirely repopulate the defective
hematopoietic system of a T mouse and rescue it from the lethal
complications of anemia (Boggs, D.R., et al., 1982, J. Clin.
Invest. 70:242-253). Doubtless, under clinical conditions in man it
would generally require more than a single stem cell to rescue the
hematopoietic system. Moreover, the presence of accessory or helper
cells (non-stem/progenitor cells that influence the growth of
stem/progenitor cells), in addition to stem and progenitor cells,
may be required (Spooncer, F., et al., 1985, Nature (London)
316:62-64), especially if the microenvironment of the host is
injured by treatments such as irradiation or chemotherapy. Thus,
while there are ways to separate hematopoietic stem and progenitor
cells from other cord blood cells (Leary, A.G., et al., 1984, J.
Clin. Invest. 74:2193-2197) and these and other methods could be
used to isolate and store pure or highly enriched preparations of
these cells for transplantation, caution should be used in attempts
at transplanting patients with purified preparations of stem and
progenitor cells.
5.1.3.2. In Vitro Cultures of Hematopoietic Stem and Progenitor
Cells
An optional re (either before or after cryopreservation) is to
expand the hematopoietic stem and progenitor cells in vitro.
However, care should be taken to ensure that growth in vitro does
not result in the production of differentiated progeny cells at the
expense of multipotent stem and progenitor cells which are
therapeutically necessary for hematopoietic reconstitution. Various
protocols have been described for the growth in vitro of cord blood
or bone marrow cells, and it is envisioned that such procedures, or
modifications thereof, may be employed (see Section 6.9 infra;
Smith, S. and Broxmeyer, H.E., 1986, Br. J. Haematol. 15 63:29-34;
Dexter, T.M., et al., 1977, J. Cell. Physiol. 91:335; Witlock, C.A.
and Witte, 0.N., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:3608-3612).
Various factors can also be tested for use in stimulation of
proliferation in vitro, including but not limited to interleukin-3
(IL-3), granulocyte-macrophage (GM)-colony stimulating factor
(CSF), IL-1 (hemopoietin-1), IL-4 (B cell growth factor), IL-6,
alone or in combination.
5.2. Cryopreservation
The freezing of cells is ordinarily destructive. 0n cooling, water
within the cell freezes. Injury then occurs by osmotic effects on
the cell membrane, cell dehydration, solute concentration, and ice
crystal formation. As ice forms outside the cell, available water
is removed from solution and withdrawn from the cell, causing
osmotic dehydration and raised solute concentration which
eventually destroy the oell. (For a discussion, see Mazur, P.,
1977, Cryobiology 14:251-272.)
These injurious effects can be circumvented by (a) use of a
cryoprotective agent, (b) control of the freezing rate, and (c)
storage at a temperature sufficiently low to minimize degradative
reactions.
Cryoprotective agents which can be used include but are not limited
to dimethyl sulfoxide (DMSO) (Lovelock, J.E. and Bishop, M.W.H.,
1959, Nature 183:1394-1395; Ashwood-Smith, M.J., 1961, Nature
190:1204-1205), glycerol, polyVinylpyrrolidine (Rinfret, A.P.,
1960, Ann. N.Y. Acad. Sci. 85:576), polyethylene glycol (Sloviter,
H.A. and Ravdin, R.G., 1962, Nature 196:548), albumin, dextran,
sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol
(Rowe, A.W., et al., 1962, Fed. Proc. 21:157), D-sorbitol,
i-inositol, D-lactose, choline chloride (Bender, M.A., et al.,
1960, J. Appl. Physiol. 15:520), amino acids (Phan The Tran and
Bender, M.A., 1960, Exp. Cell Res. 20:651), methanol, acetamide,
glycerol monoacetate (Lovelock, J.E., 1954, Biochem. J. 56:265),
and inorganic salts (Phan The Tran and Bender, M.A., 1960, Proc.
Soc. Exp. Biol. Med. 104:388; Phan The Tran and Bender, M.A., 1961,
in Radiobiology, Proceedings of the Third Australian Conference on
Radiobiology, Ilbery, P.L.T., ed., Butterworth, London, p. 59). In
a preferred embodiment, DMSO is used, a liquid which is nontoxic to
cells in low concentration. Being a small molecule, DMSO freely
permeates the cell and protects intracellular organelles by
combining with water to modify its freezability and prevent damage
from ice formation. Addition of plasma (e.g., to a concentration of
20-25%) can augment the protective effect of DMSO. After addition
of DMSO, cells should be kept at 0.degree. C. until freezing, since
DMSO concentrations of about 1% are toxic at temperatures above
4.degree. C.
A controlled slow cooling rate is critical. Different
cryoprotective agents (Rapatz, G., et al., 1968, Cryobiology
5(1):18-25) and different cell types have different optimal cooling
rates (see =.g . Rowe, A.W. and Rinfret, A.P., 1962, Blood 20:636;
RoWe, A.W., 1966, Cryobiology 3(1):12-18; Lewis, J.P., et al.,
1967, Transfusion 7(1):17-32; and Mazur, P., 1970, Science
168:939-949 for effects of cooling velocity on survival of
marrow-stem cells and on their transplantation potential). The heat
of fusion phase where water turns to ice should be minimal. The
cooling procedure can be carried out by use of, e.g., a
programmable freezing device or a methanol bath procedure.
Programmable freezing apparatuses allow determination of optimal
cooling rates and facilitate standard reproducible cooling.
Programmable controlled-rate freezers such as Cryomed or Planar
permit tuning of the freezing regimen to the desired cooling rate
curve. For example, for marrow cells in 10% DMSO and 20% plasma,
the optimal rate is 1 to 3.degree. C./minute from 0.degree. C. to
-80.degree. C. In a preferred embodiment, this cooling rate can be
used for the neonatal cells of the invention. The container holding
the cells must be stable at cryogenic temperatures and allow for
rapid heat transfer for effective control of both freezing and
thawing. Sealed plastic vials (e.g., Nunc, Wheaton cryules) or
glass ampules can be used for multiple small amounts (1-2 ml),
while larger volumes (100-200 ml) can be frozen in polyolefin bags
(e.g., Delmed) held between metal plates for better heat transfer
during cooling. (Bags of bone marrow cells have been successfully
frozen by placing them in -80.C freezers which, fortuitously, gives
a cooling rate of approximately 3.degree. C./minute).
In an alternative embodiment, the methanol bath method of cooling
can be used. The methanol bath method is well-suited to routine
cryopreservation of multiple small items on a large scale. The
method does not require manual control of the freezing rate nor a
recorder to monitor the rate. In a preferred aspect, DMSO-treated
cells are precooled on ice and transferred to a tray containing
chilled methanol which is placed, in turn, in a mechanical
refrigerator (e.g., Harris or Revco) at -80.degree. C. Thermocouple
measurements of the methanol bath and the samples indicate the
desired cooling rate of 1 to 3.degree. C./minute. After at least
two hours, the specimens have reached a temperature of -80.degree.
C. and can be placed directly into liquid nitrogen (-196.degree.
C.) for permanent storage.
After thorough freezing, cells can be rapidly transferred to a
long-term cryogenic storage vessel. In a preferred embodiment,
samples can be cryogenically stored in liquid nitrogen
(-196.degree. C.) or its vapor (-165.degree. C.). Such storage is
greatly facilitated by the availability of highly efficient liquid
nitrogen refrigerators, which resemble large Thermos containers
with an extremely low vacuum and internal super insulation, such
that heat leakage and nitrogen losses are kept to an absolute
minimum.
In a particular embodiment, the cryopreservation procedure
described in Section 6.4 infra is envisioned for use. The
sterilized storage cryules preferably have their caps threaded
inside, allowing easy handling without contamination. Suitable
racking systems are commercially available and can be used for
cataloguing, storage, and retrieval of individual specimens.
Considerations and procedures for the manipulation,
cryopreservation, and long term storage of hematopoietic stem
cells, particularly from bone marrow or peripheral blood, is
largely applicable to the neonatal and fetal stem cells of the
invention. Such a discussion can be found, for example, in the
following references, incorporated by reference
herein: Gorin, N.C., 1986, Clinics In Haematology 15(1):19-48;
Bone-Marrow Conservation, Culture and Transplantation, Proceedings
of a Panel, Moscow, July 22-26, 1968, International Atomic Energy
Agency, Vienna, pp. 107-186.
Other methods of cryopreservation of viable cells, or modifications
thereof, are available and envisioned for use (e.g., cold
metal-mirror techniques; Livesey, S.A. and Linner, J.G., 1987,
Nature 327:255; Linner, J.G., et al., 1986, J. Histochem. Cytochem.
34(9):1123-1135; see also U.S. Pat. No. 4,199,022 by Senkan et al.,
U.S. Pat. No. 3,753,357 by Schwartz, U.S. Pat. No. 4,559,298 by
Fahy; and Section 2.3, supra).
5.3. Recovering Stem and Progenitor Cells from the Frozen State
5.3.1. Thawing
Frozen cells are preferably thawed quickly (e.g., in a water bath
maintained at 37-41.degree. C.) and chilled immediately upon
thawing. In particular, the vial containing the frozen cells can be
immersed up to its neck in a warm water bath; gentle rotation will
ensure mixing of the cell suspension as it thaws and increase heat
transfer from the warm water to the internal ice mass. As soon as
the ice has completely melted, the vial can be immediately placed
in ice (see Section 6.5, infra).
5.3.2. Optional Procedures
In a preferred embodiment of the invention, the neonatal blood
sample as thawed can be infused for hematopoietic reconstitution.
Thus, it is envisioned that whole neonatal blood, cryopreserved and
thawed, can be infused for therapy. However, several procedures,
relating to processing of the thawed cells are available, and can
be employed if deemed desirable. Such procedures are discussed
infra.
It may be desirable to treat the cells in order to prevent cellular
clumping upon thawing. To prevent clumping, various procedures can
be used, including but not limited to, the addition before and/or
after freezing of DNase (Spitzer, G., et al., 1980, Cancer
45:3075-3085), low molecular weight dextran and citrate,
hydroxyethyl starch (Stiff, P.J., et al., 1983, Cryobiology
20:17-24), etc.
The cryoprotective agent, if toxic in humans, should be removed
prior to therapeutic use of the thawed neonatal stem and progenitor
cells. In an embodiment employing DMSO as the cryopreservative, it
is preferable to omit this step in order to avoid cell loss, since
DMSO has no serious toxicity. However, where removal of the
cryoprotective agent is desired, the removal is preferably
accomplished upon thawing.
One way in which to remove the cryoprotective agent is by dilution
to an insignificant concentration. This can be accomplished by
addition of medium, followed by, if necessary, one or more cycles
of centrifugation to pellet cells, removal of the supernatant, and
resuspension of the cells. For example, intracellular DMSO in the
thawed cells can be reduced to a level (less than 1%) that will not
adversely affect the recovered cells. This is preferably done
slowly to minimize potentially damaging osmotic gradients that
occur during DMSO removal (see Section 6.5, infra.)
After removal of the cryoprotective agent, cell count (e.g., by use
of a hemocytometer) and viability testing (e.g., by trypan blue
exclusion; Kuchler, R.J. 1977, Biochemical Methods in Cell Culture
and Virology, Dowden, Hutchinson & Ross, Stroudsburg, Pa., pp.
18-19; 1964, Methods in Medical Research, Eisen, H.N., et al.,
eds., Vol. 10, Year Book Medical Publishers, Inc., Chicago, pp.
39-47) can be done to confirm cell survival.
Other procedures which can be used, relating to processing of the
thawed cells, include enrichment for hematopoietic stem and
progenitor cells (see Section 5.1.3.1, supra) and expansion by in
vitro culture (see Section 5.1.3.2, supra). However, in a preferred
embodiment, these steps can be omitted in order to minimize cell
loss.
5.4. Examination of Cells Recovered for Clinical Therapy
In a preferred, but not required, aspect of the invention, thawed
cells are tested by standard assays of viability (e.g., trypan blue
exclusion) and of microbial sterility (see Section 5.1.2, supra),
and tested to confirm and/or determine their identity relative to
the patient, and for hematopoietic function.
5.4.1. Identity Testing
Methods for identity testing which can be used include but are not
limited to HLA (the major histocompatibility complex in man) typing
(Bodmer, W., 1973, in Manual of Tissue Typing Techniques, Ray,
J.G., et al., eds., DHEW Publication No. (NIH) 74-545, pp. 24-27),
and DNA fingerprinting, which can be used to establish the genetic
identity of the cells. DNA fingerprinting (Jeffreys, A.J., et al.,
1985, Nature 314:67-73) exploits the extensive restriction fragment
length polymorphism associated with hypervariable minisatellite
regions of human DNA, to enable identification of the origin of a
DNA sample, specific to each individual (Jeffreys, A.J., et al.,
1985, Nature 316:76; Gill, P., et al., 1985, Nature 318:577;
Vassart, G., et al., 1987, Science 235:683), and is thus preferred
for use.
In a specific embodiment of the invention in which the cells
recovered for therapy are to be used in an autologous system, the
cells should match exactly the recipient patient from whom they
originally came.
5.4.2. Assays For Stem and Progenitor Cells
Any of numerous assays for hematopoietic stem or progenitor cells
may be used (see Section 2.1). Examples of specific assays are
described in Section 6.6 and subsections, infra. Modifications of
the assays therein described are also envisioned for use. For
example, various factors, alone or in combination, can be tested
for stimulation of colony formation upon inclusion in the culture
mixture (see Broxmeyer, H.E., 1986, Int. J. Cell Cloning 4:378-405;
Lu, L. and Broxmeyer, H.E., 1983, Exp. Hematol. 11(8):721-729; Lu,
L. and Broxmeyer, H.E., 1985, Exp. Hematol. 13:989-993); such
factors include but are not limited to oxygen tension, E-type
prostaglandins, interleukin-3 (IL-3), granulocyte-macrophage
(GM)-colony stimulating factor (CSF), granulocyte (G)-CSF,
macrophage (M)-CSF (CSF-1), erythropoietin, IL-1, IL-4 (B cell
growth factor), hemin (ferric chloride protoporphyrin IX), and
media conditioned by various cell types. Culture assay methods may
thus be changed to employ more efficient conditions for colony
growth. In addition to in vitro colony forming assays, a stem cell
assay for CFU-S (colony forming unit-spleen) can be done. In this
assay, cells considered to be multipotential stem cells with
self-renewal capacity can be measured by counting the number of
colonies (nodules) on the spleen(s) of lethally-irradiated mice
that have been inoculated with a composition containing the
cells.
In a particular embodiment, low density Ficoll-Hypaque-separated
cells (density less than 1.077 gm/cm.sup.3), which include the stem
and progenitor cells, are plated, usually 0.5-2.0.times.10.sup.5
per plate, for recognition of S (stem) cells, and progenitor cells
of the CFU-GEMM (multipotent) and CFU-GM and BFU-E (more
differentiated) categories.
5.5 Hematopoietic Reconstitution
The neonatal hematopoietic stem and progenitor cells of the present
invention can be used therapeutically for hematopoietic
reconstitution, with either syngeneic or allogeneic hosts. The
neonatal cells can be introduced into a patient for repopulation of
the blood and other hematopoietic organs in the treatment or
prevention of various diseases or disorders, as described infra in
Section 5.6. Introduction of the neonatal cells can occur by any
method known in the art, with systemic infusion of cells being the
preferred route.
In a preferred embodiment of the invention, the neonatal cells are
autologous (self) cells, i.e., the cells were originally derived
from the host recipient. Such an embodiment avoids the
immunosuppressive regimens (e.g., irradiation, chemotherapy) which
are often necessary in allogeneic transplants in order to avoid
debilitating graft versus host or host versus graft disease.
5.6. Therapeutic Uses
Reconstitution of the hematopoietic system (or immune system) with
the neonatal stem and progenitor cells of the present invention can
be therapeutically valuable for a large number of diseases and
disorders.
In a preferred embodiment involving the use of autologous neonatal
cells, the infusion of previously cryopreserved neonatal
hematopoietic stem and progenitor cells for hematopoietic
reconstitution at any time after birth can not only be applied in
the treatment of diseases which are presently known to be curable
by allogeneic bone marrow transplantation, but also offers
therapeutic potential for a number of additional diseases which
presently are not considered likely to benefit from allogeneic
marrow transplantation. This is due to the fact that allogeneic
marrow transplantation (except for the few patients who are already
immunologically incompetent) requires pretransplantation
conditioning of the recipient with intensive cytoreduction with
irradiation or chemotherapy for the purpose of eliminating the host
(recipient) immune system in order to allow the transplanted marrow
cells to engraft. This pretransplantation cytoreduction in
combination with allogeneic HLA-identical marrow transplantation
can result in a number of serious transplantation-induced
complications such as life-threatening infections, long-lasting
immunodeficiencies, and frequently, graft-versus-host disease.
Disorders that can be treated by infusion of stem cells include but
are not limited to five broad categories. First are diseases
resulting from a failure or dysfunction of normal blood cell
production and maturation (i.e., aplastic anemia and
hypoproliferative stem cell disorders). The second group are
neoplastic, malignant diseases in the hematopoietic organs (e.g.,
leukemia and lymphomas). The third group of disorders comprises
those of patients with a broad spectrum of malignant solid tumors
of non-hematopoietic origin. Stem cell infusion in these patients
serves as a bone marrow rescue procedure, which is provided to a
patient following otherwise lethal chemotherapy or irradiation of
the malignant tumor. The fourth group of diseases consists of
autoimmune conditions, where the stem cells serve as a source of
replacement of an abnormal immune system. The fifth group of
diseases comprises a number of genetic disorders which can be
corrected by infusion of hematopoietic stem cells, preferably
syngeneic, which prior to transplantation have undergone gene
therapy. Particular diseases and disorders which can be treated by
hematopoietic reconstitution with neonatal stem and progenitor
cells include but are not limited to those listed in Table II, and
described infra.
TABLE II ______________________________________ DISEASES OR
DISORDERS WHICH CAN BE TREATED BY HEMATOPOIETIC RECONSTITUTION WITH
NEONATAL STEM AND PROGENITOR CELLS
______________________________________ I. Diseases resulting from a
failure or dysfunction of normal blood cell production and
maturation hyperproliferative stem cell disorders aplastic anemia
pancytopenia agranulocytosis thrombocytopenia red cell aplasia
Blackfan-Diamond syndrome due to drugs, radiation, or infection
idiopathic II. Hematopoietic malignancies acute lymphoblastic
(lymphocytic) leukemia chronic lymphocytic leukemia acute
myelogenous leukemia chronic myelogenous leukemia acute malignant
myelosclerosis multiple myeloma polycythemia vera agnogenic
myelometaplasia Waldenstrom's macroglobulinemia Hodgkin's lymphoma
non-Hodgkins's lymphoma III. Immunosuppression in patients with
malignant, solid tumors malignant melanoma carcinoma of the stomach
ovarian carcinoma breast carcinoma small cell lung carcinoma
retinoblastoma testicular carcinoma glioblastoma rhabdomyosarcoma
neuroblastoma Ewing's sarcoma lymphoma IV. Autoimmune diseases
rheumatoid arthritis diabetes type I chronic hepatitis multiple
sclerosis systemic lupus erythematosus V. Genetic (congenital)
disorders anemias familial aplastic Fanconi's syndrome Bloom's
syndrome pure red cell aplasia (PRCA) dyskeratosis congenita
Blackfan-Diamond syndrome congenital dyserythropoietic syndromes
I-IV Chwachmann-Diamond syndrome dihydrofolate reductase
deficiencies formamino transferase deficiency Lesch-Nyhan syndrome
congenital spherocytosis congenital elliptocytosis congenital
stomatocytosis congenital Rh null disease paroxysmal nocturnal
hemoglobinuria G6PD (glucose-6-phosphate dehydrogenase) variants
1,2,3 pyruvate kinase deficiency congenital erythropoietin
sensitivity deficiency sickle cell disease and trait thalassemia
alpha, beta, gamma met-hemoglobinemia congenital disorders of
immunity severe combined immunodeficiency disease (SCID) bare
lymphocyte syndrome ionophore-responsive combined immunodeficiency
combined immunodeficiency with a capping abnormality nucleoside
phosphorylase deficiency granulocyte actin deficiency infantile
agranulocytosis Gaucher's disease adenosine deaminase deficiency
Kostmann's syndrome reticular dysgenesis congenital leukocyte
dysfunction syndromes VI. Others osteopetrosis myelosclerosis
acquired hemolytic anemias acquired immunodeficiencies infectious
disorders causing primary or secondary immunodeficiencies bacterial
infections (e.g., Brucellosis, Listerosis, tuberculosis, leprosy)
parasitic infections (e.g., malaria, Leishmaniasis) fungal
infections disorders involving disproportions in lymphoid cell sets
and impaired immune functions due to aging phagocyte disorders
Kostmann's agranulocytosis chronic granulomatous disease
Chediak-Higachi syndrome neutrophil actin deficiency neutrophil
membrane GP-180 deficiency metabolic storage diseases
mucopolysaccharidoses mucolipidoses miscellaneous disorders
involving immune mechanisms Wiskott-Aldrich Syndrome alpha
1-antitrypsin deficiency ______________________________________
5.6.1. Diseases Resulting From a Failure or Dysfunction of Normal
Blood Cell Production and Maturation
In this embodiment of the invention, reconstitution of the
hematopoietic system with neonatal stem and progenitor cells can be
used to treat diseases resulting from a failure or dysfunction of
normal blood cell production and maturation, i.e., aplastic anemia
and hypoproliferative stem cell disorders. These disorders entail
failure of stem cells in bone marrow to provide normal numbers of
functional blood cells. The aplastic anemias result from the
failure of stem cells to give rise to the intermediate and mature
forms of red cells, white cells, and platelets. Red cell production
is usually most seriously affected, but a marked decrease in
production of other mature blood cell elements is also seen. The
large majority of these anemias are acquired during adult life, and
do not have any apparent genetic predisposition. About half of
these acquired anemias arise in the absence of any obvious
causative factor such as exposure to poisons, drugs or disease
processes that impair stem cell function; these are termed
idiopathic aplastic anemias. The remaining cases are associated
with exposure to an extremely diverse array of chemicals and drugs
and can also occur as the consequence of viral infections such as
hepatitis, and after pregnancy. Other types of aplastic anemia are
termed agranulocytosis or thrombocytopenia to indicate that the
major deficiency lies in particular white cells or in platelet
production, respectively. Agranulocytosis may be associated with
autoimmune syndromes such as systemic lupus erythematosis (SLE) or
with infections, particularly neonatal rubella.
The overall mortality of all patients with aplastic anemias, in the
absence of stem cell therapy, is high. Approximately 60-75% of
individuals suffering from the disorder die within 12 months, in
the absence of new stem cells. The overall incidence of these
diseases is approximately 25 new cases per million persons per
year. Although it is extremely unlikely that a single pathogenic
mechanism accounts for all aplastic anemias, it is clear that
provision of new hematopoietic stem cells is usually sufficient to
allow permanent recovery, since transplantation of patients with
aplastic anemia with bone marrow obtained from identical twins
(i.e., syngeneic) (Pillow, R.P., et al., 1966, N. Engl. J. Med.
275(2):94-97) or from HLA-identical siblings (i.e., allogeneic)
(Thomas, E.D., et al., Feb. 5, 1972, The Lancet, pp. 284-289) can
fully correct the disease. However, some patients with aplastic
anemia reject the transplanted marrow. This complication is
particularly common among patients who have been immunologically
sensitized as a result of multiple therapeutic blood transfusions.
In a preferred embodiment of the invention employing autologous
neonatal stem cells for hematopoietic reconstitution, such a
complication can be avoided.
In a specific embodiment of the invention, hematopoietic
reconstitution by infusion of neonatal stem cells can be used for
the treatment of Fanconi's anemia, an autosomal recessive disease
exhibited by congenital malformations associated with bone marrow
failure. The stem cell defect is associated with chromosomal
instability, and increased risk for malignancy. The disease is
always fatal in its natural course. This embodiment of the
invention is illustrated by way of example in Section 12, infra,
which describes the infusion of neonatal blood comprising
hematopoietic stem and progenitor cells into a patient with
Fanconi's anemia for treatment of the disease. In a preferred
aspect of this embodiment, the patient is conditioned before stem
cell infusion, by a conditioning regimen which is modified
according to cell sensitivity to alkylating agents and to
irradiation (see Gluckman, E., et al., 1983, Brit. J. Haematol.
54:431-440; Gluckman, E., et al., 1984, Seminars in Haematol.
21(1):20-26; Gluckman, E. and Dutreix, J., 1985, The Cancer
Bulletin 37(5):238-242; Gluckman, E., et al., 1980, Brit. J.
Haematol. 45:557- 564; all incorporated by reference herein). For
example, cytogenetic analysis can be used to predict cell
sensitivity to alkylating agents (Berger, R., et al., 1980, Brit.
J. Haematol. 45:565-568). Tests for radiosensitivity have also been
described (Gluckman, E. and Dutreix, J., 1985, The Cancer Bulletin
37(5):238-242; Gluckman, E., et al., 1983, Brit. J. Haematol.
54:431-440). In a particular embodiment, a conditioning regimen
using cyclophoshamide and thoraco-abdominal irradiation can be
employed.
5.6.2. Hematopoietic Malignancies
Hyperproliferative malignant stem cell disorders which can be
treated by hematopoietic reconstitution with neonatal stem and
progenitor cells include but are not limited to acute lymphocytic
leukemia, chronic lymphocytic leukemia, acute and chronic
myelogenous leukemia, multiple myelomas, polycythemia vera,
agnogenic myelometaplasia, Waldenstrom,s macroglobulinemia, and
Hodgkins and non-Hodgkins lymphoma. These leukemias are currently
treated by chemotherapy and, when feasible, allogeneic bone marrow
transplantation. However, allogeneic HLA identical sibling bone
marrow is available only to less than one-third of patients, and
this treatment is associated with transplantation-related
complications such as immunodeficiency and graft versus host
disease. Provision of syngeneic (self) cryopreserved hematopoietic
stem cells, according to a preferred embodiment of the invention,
would permit hematopoietic reconstitution of patients lacking
suitable allogeneic donors and eliminate the risks of graft versus
host disease arising from allogeneic marrow transplantation.
5.6.3. Malignant, Solid Tumors of Non-Hematopoietic Origin
Hematopoietic reconstitution can greatly aid in the treatment of
patients with malignant, solid tumors undergoing irradiation or
chemotherapy, by providing new stem cells. Such tumors include but
are not limited to those listed in Table II, supra.
There is increasing evidence that a number of cancers are
remarkably sensitive to extremely high doses of normally
ineffective anti-neoplastic drugs. These cancers include malignant
melanoma, carcinomas of the stomach, ovary, and breast, small cell
carcinoma of the lung, and malignant tumors of childhood (including
retinoblastoma and testicular carcinoma), as well as certain brain
tumors, particularly glioblastoma. However, such intensive high
dose chemotherapy is not widely used because it frequently causes
hematopoietic failure and death. The provision of new stem cells
after intensive chemotherapy has been accomplished by using bone
marrow cells obtained from patients before administration of the
cytotoxic drugs (Spitzer, G., et al., 1980, Cancer 45:3075-3085).
This approach has two major difficulties. First, it has not been
routinely possible to obtain sufficient numbers of bone marrow
cells from chronically ill patients with cancer. In addition,
clinicians have been reluctant to use this approach because of the
probability that the patient's bone marrow cells are contaminated
by small numbers of neoplastic cells. This is particularly true in
the hematologic malignancies, but also pertains to most metastatic
cancers. The provision of stem cells according to the present
invention, obtained at a time of health, before the onset of
cancer, can permit the use of potentially curative intensive
chemotherapy without the risk of stem cell failure.
5.6.4. Autoimmune Disorders
Many chronic inflammatory and degenerative diseases are
characterized by a continuous immune reaction against the body's
own tissues. Such autoimmune disorders include but are not limited
to rheumatoid arthritis and other inflammatory osteopathies,
diabetes type I, chronic hepatitis, multiple sclerosis, and
systemic lupus erythematosus. Autoimmune disorders are often
treated by lymphoid irradiation. Use of the neonatal hematopoietic
stem and progenitor cells for hematopoietic reconstitution
according to the present invention can be extremely valuable after
radiotherapy.
Anti-inflammatory drugs such as steroids retard the inflammatory
cells which are activated by autoreactive T cells, but do not
prevent T cells which recognize self-proteins from activating new
inflammatory cells. A more direct approach to treating autoimmune
diseases depends on eradication of T cells by irradiation of the
lymphoid tissues, and relying on stem cells from the unirradiated
bone marrow to repopulate the patient's hematopoietic system. The
rationale is that the formation of new populations of mature T
cells from bone marrow stem cells may result in absence of T cells
that have reactivity to self-specific antigens. This procedure,
called total lymphoid irradiation (TLI), has been used to treat
intractable rheumatoid arthritis (Strober, S., et al., 1985, Annals
of Internal Medicine 102:441-449, 450-458). These clinical trials
showed that in the majority of otherwise intractable cases, joint
disease was significantly alleviated for at least 2-3 years.
However, the major drawback to such treatment is failure of stem
cells in the bone marrow of these elderly patients to efficiently
repopulate the hematopoietic system, resulting in infections and
bleeding disorders. Analogous studies have been made of the effects
of TLI as an alternative to cytotoxic drugs for treatment of SLE
(Strober, S., et al., 1985, Ann. Internal Med. 102:450). Studies of
the use of TLI to treat intractable SLE have also shown that this
treatment alleviates disease activity, but is severely limited by
failure of bone marrow stem cells to rapidly and efficiently
repopulate the hematopoietic system after irradiation. In a
preferred aspect of the invention, the availability of an
individual's own stem cells, obtained at birth, can allow efficient
repopulation of mature T cells in an adult environment, after
minimal lymphoid radiotherapy, and can thus render this therapy
significantly more effective.
5.6.5. Gene Therapy
Hematopoietic reconstitution with the neonatal stem and progenitor
cells of the invention which have undergone gene therapy, i.e.,
which have stably incorporated a heterologous gene capable of
expression by their progeny cells, can be of great value in the
treatment of diseases and disorders affecting cells of
hematopoietic lineage. In one embodiment, hematopoietic
reconstitution with such recombinant stem cells can be used in the
treatment of genetic disorders of the hematopoietic system. Such
genetic disorders include but are not limited to those listed in
Table II, supra. Genetic deficiencies or dysfunctions of
hematopoietic cells can be treated by supplying, to a patient,
recombinant stem and progenitor cells. In a specific embodiment,
patients who have hematopoietic cells which lack a gene or have a
mutant gene, can be reconstituted with neonatal stem and progenitor
cells that have incorporated a functional counterpart of the
deficient gene. In particular, such genes which can be subject to
gene therapy include but are not limited to hemoglobin or enzymes
which mediate its synthetic pathway (e.g., for treatment of anemias
such as beta-thalassemia, sickle-cell disease).
In another specific embodiment, patients with infections by
pathogenic microorganisms which occur in or affect a hematopoietic
cell lineage can be treated with recombinant neonatal stem and
progenitor cells. Such recombinant stem and progenitors can contain
a heterologous gene which is expressed as a product which
ameliorates disease symptoms, is toxic to the pathogen without
significant detriment to the host, or interferes with the
pathogen's life cycle, etc. Pathogens which cause infections which
may be treated with recombinant stem cells according to this
embodiment of the invention include but are not limited to
lymphotropic viruses such as Human Immunodeficiency Virus (HIV, the
etiological agent of acquired immune deficiency symdrome (AIDS))
(Gallo et al., 1984, Science 224:500-503; Barre-Sinoussi, F., et
al., 1983, Science 220:868; Levy, J.A., et al., 1984, Science
225:840); gram-negative bacilli such as Brucella or Listeria; the
mycobacterium which cause tuberculosis, or which cause Hansen's
disease (leprosy); parasites such as Plasmodium (the etiological
agents of malaria), or Leishmania; and fungi (such as those that
cause pneumonia and other lethal infections secondary to
immunodeficiencies) (for a discussion of many of these disorders,
see Harrison's Principles of Internal Medicine, 1970, 6th Edition,
Wintrobe, M.M., et al., eds., McGraw-Hill, New York, pp. 798-1044).
As a particular embodiment, it is possible to construct recombinant
neonatal stem or progenitor cells that express a sequence which is
"anti-sense" to the nucleic acid of a hematopoietic cell pathogen.
Such a sequence, which is complementary to the pathogen's RNA or
DNA, can hybridize to and inactivate such RNA or DNA, inhibiting
the function or expression of the nucleic acid and disrupting the
pathogen's life cycle. As a particular example, recombinant
neonatal hematopoietic cells can be used in the treatment of AIDS,
a disorder which is caused by HIV, apparently by infection of
T4.sup.+ lymphocytes (Dagleish et al., 1984, Nature 312:763-766;
Klatzmann et al., 1984, Nature 312:767-768). Recombinant neonatal
stem and progenitor cells which express an anti-sense nucleic acid
that is complementary to a critical region (e.g., the long-terminal
repeat or polymerase sequence ) of the HIV genome (Wain-Hobson et
al., 1985, Cell 40:9-17) can be used for hematopoietic
reconstitution for the treatment of AIDS.
Numerous techniques are known in the art for the introduction of
foreign genes into cells and may be used to construct the
recombinant neonatal hemapoietic stem and progenitor cells for
purposes of gene therapy, in accordance with this embodiment of the
invention. The technique used should provide for the stable
transfer of the heterologous gene sequence to the stem cell, so
that the heterologous gene sequence is heritable and expressible by
stem cell progeny, and so that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
Techniques which may be used include but are not limited to
chromosome transfer (e.g., cell fusion, chromosome-mediated gene
transfer, micro cell-mediated gene transfer), physical methods
(e.g., transfection, spheroplast fusion, microinjection,
electroporation, liposome carrier), viral vector transfer (e.g.,
recombinant DNA viruses, recombinant RNA viruses) etc. (described
in Cline, M.J., 1985, Pharmac. Ther. 29:69-92, incorporated by
reference herein).
5.6.6. Miscellaneous Disorders Involving Immune Mechanisms
Hematopoietic reconstitution with the neonatal hematopoietic stem
and progenitor cells of the present invention can be used to treat
patients with various miscellaneous disorders involving immune
mechanisms. Disorders resulting from inefficient function, lack of
function, or dysfunction, of an hematopoietic cell lineage can be
alleviated by replacement of the hematopoietic cell progeny with
those derived from neonatal stem and progenitor cells of normal
function. In a specific embodiment, a hemolytic disorder can be
treated (for a discussion of hemolytic disorders, see e.g., 1985,
Cecil, Textbook of Medicine, Wyngaarden, J.B. and Smith, L.H.,
eds., 17th Ed., W.B. Saunders Co., pp. 900-915). Hemolytic
disorders acquired during adult life account for the large majority
of this form of anemia, and reflect the destruction of red cells by
lymphocyte products. Stem cell replacement therapy with the
neonatal cells of the invention can provide a new source of red
cells, and, in an embodiment employing autologous cells, can
replace destructive lymphocytes with newly formed cells which are
unlikely to generate an immune response against the recipient's red
cells. In another specific embodiment, patients whose immune system
is compromised e.g., as a result of irradiation or chemotherapy,
can be treated by hematopoietic reconstitution with neonatal
hemapoietic stem and progenitor cells (see Section 5.6.3). In yet
another embodiment, disorders involving disproportions in lymphoid
cell sets and impaired immune functions due to aging can be treated
by reconstitution with the neonatal cells of the invention. Genetic
disorders of metabolism which result in pathologic accumulations of
metabolic products in the marrow (e.g., osteopetrosis, metabolic
storage diseases) are also among the many disorders envisioned for
treatment.
In addition, immune deficiencies which are the primary or secondary
result of infection by pathogenic microorganisms can be treated by
hematopoietic reconstitution with the stem cells of the invention.
In this embodiment, neonatal stem cells can serve as a source of
cells of the hematopoietic cell lineage which are needed by the
patient. For example, immune deficiencies caused by microorganisms
which are intracellular pathogens of hematopoietic cells, can be
treated by the provision of new hematopoietic cells, supplied by
infused neonatal stem cells. Microorganisms causing immune
deficiencies which may be treated according to this embodiment of
the invention include but are not limited to gram-negative bacilli
such as Brucella or Listeria, the mycobacterium which are the
etiological agents of tuberculosis or of Hansen's disease
(leprosy), parasites such as Plasmodium (the etiological agents of
malaria) or Leishmania, and fungi (such as those that cause
pneumonia and other lethal infections secondary to
immunodeficiencies) (for a discussion of many of these disorders,
see Harrison's Principles of Internal Medicine, 1970, 6th Edition,
Wintrobe, M.M., et al., eds., McGraw-Hill, New York, pp.
798-1044).
5.7 Generation and Use of Hematopoietic Stem and Progenitor Cell
Progeny
In another method of the invention, progeny cells of hematopoietic
stem and progenitor cells of fetal or neonatal blood can be
generated in vitro; the differentiated progeny cells thus generated
can be therapeutically useful. For example, in one embodiment of
this aspect of the invention, hematopoietic stem cells and/or
CFU-GEMM progenitor cells, before or after cryopreservation and
thawing, can be induced to differentiate into platelets. Such
platelets can be used, for example, for infusion into a patient
with thrombocytopenia. In another embodiment, granulocytes can be
generated in vitro prior to infusion into a patient. One or more of
the hematopoietic progeny cells can be generated in vitro, allowing
for the in vitro production of blood components. In a preferred
embodiment, the generation of differentiated blood components is
accompanied by expansion of the hematopoietic stem and progenitor
cell pool, in order to allow for production of a greater quantity
of differentiated cells. Various growth factors can be used to
promote expansion and/or differentiation of hematopoietic stem and
progenitor cells, such as cytokines (growth factors) including but
not limited to G-CSF, CSF-1, IL-3, IL-5, tumor necrosis
factor-.alpha., and .gamma.-interferon. The blood components which
are thus produced have uses which are not limited to therapeutic
uses in vivo. For example, such progeny cells can be used in vitro,
e.g., for the production and isolation of hematopoietic cell
products such as growth factors, antibodies, etc.
6. EXAMPLES
6.1. Collection of Human Umbilical Cord and Placental Blood
Neonatal blood was collected from human umbilical cords by gravity
drainage and/or by needle aspiration from delivered placentas. Data
for the volumes obtained in one series of collections from
individual births is shown in FIG. 1, and demonstrates that volumes
of 50 ml or more can be obtained. Data from another series of
collections is shown in FIG. 2, with the collections from
individual births identified by method of collection and delivery
type, as either: gravity flow, vaginal delivery; gravity flow,
Caesarian section; placental aspiration, vaginal delivery; or
placental aspiration, Caesarian section. The data show that the
majority of the collections had a total volume of greater than 30
ml although many contained less than 50 ml. In recent collections,
we have been able to obtain volumes approximately twice as large as
shown in FIG. 2 (e.g., 99 ml blood from a neonate, after a 36 week
gestation) by using needle aspirations from the delivered placenta,
at the root of the placenta and in the distended surface veins,
combined with cord drainage.
Cord blood collections were done essentially as described in
Section 5.1.1 and subsections, supra, and as detailed infra.
Cord blood collection kits consisted of:
wide-mouth bottle (200 ml) (Corning, Corning, NY), Cat. No.
25625-200; VWR, South Plainfield, NJ, Cat. No. 28199-756)
wrap (operating room drape sheet)
For collections by needle aspiration, 60 cc syringes B-D Luerlok
(VWR, Cat. No. BD5663) and 18 gauge needles 1 1/2 inch (VWR, Cat.
No. BD5196) were used.
Collection bottles were sterilized before collection by
beta-irradiation with 2.5 megarads from a tungsten source
(Dynamatron Accelerator, Radiation Dynamics, Inc., Melville, New
York). Syringes and needles were autoclaved. (Alternatively, the
syringes and needles were sterilized with ethylene oxide.)
Twenty ml of CPD (citrate-phosphate-dextrose) was added to each
cord blood collection container, as an anticoagulent. CPD was
prepared according to the following:
______________________________________ Trisodium citrate
(dihydrate) 28.8 g Citric acid (monohydrate) 3.2 g Sodium
dihydrogen phosphate 2.19 g (monohydrate) Dextrose 25.0 g
______________________________________
Bring volume to 1,000 ml; pH should be 5.63. Use at 20 ml CPD per
up to approximately 120 ml blood.
In selected samples, acid-citrate-dextrose (ACD) (Hurn, B.A.L.,
1968, Storage of Blood, Academic Press, New York, p. 137) was used
instead of CPD. ACD was prepared according to the following:
______________________________________ Trisodium citrate
(dihydrate) 1.65 g Citric acid (monohydrate) 1.983 g Dextrose
(anhydrous) 6.13 g in a total volume of 250 ml
______________________________________
The substitution of ACD for CPD caused no observable differences in
the hematopoietic stem and progenitor cell counts which were
obtained.
Penicillin and streptomycin were also added to the collected blood.
0.01 .times. cord blood volume, of a solution consisting of 5000
units penicillin per ml and 500 ug streptomycin per ml, was added
to each cord blood sample.
Approximately 109 of the human umbilical cord blood samples which
were collected were subjected to further analysis as described
infra.
6.2. Hematopoietic Stem and Progenitor Cells in Collected Cord
Blood
The approximately 109 collected cord blood samples of section 6.1
were sent by overnight mail (in polystyrene mailers; Fisher,
Fairhaven, New Jersey, Cat. No. 03-528-10) to a processing site
where they were separated, counted for viable cell numbers, set up
for hematopoietic progenitor cell assays (in most cases), frozen
away for storage, and in some cases defrosted for assessment of
recovery of total nucleated cells and hematopoietic progenitors
(see Section 6.7, infra). The progenitor cells evaluated included
immature and mature granulocyte-macrophage (day 7 CFU-GM, day 14
CFU-GM), "mmature" and "mature" erythroid (BFU-E-1, BFU-E-2), and
multipotential cells (see Section 6.6 and subsections, infra for
assays of progenitor cells). Table III presents a complete list of
the samples received and the numbers of hematopoietic progenitor
cells per sample present in the low density fraction after
separation with Ficoll-Hypaque.
TABLE III
__________________________________________________________________________
HEMATOPOIETIC PROGENITOR CELLS IN HUMAN CORD BLOOD Total Viable
Viable Total Low Low Nucleated Density Density Total Progenitor
Cells Total Blood Blood Blood (Expressed as Colonies .times.
10.sup.-3) Volume Cells Cells Cells CFU-GM CFU-GM CFU- Sample (ml)
(.times. 10.sup.-6) (%) (.times. 10.sup.-6) (Day 7) (Day 14)
BFU-E-2 BFU-E-1 GEMM Comments
__________________________________________________________________________
CB-1 72.5 631 93.4 112 40 159 116 76 90 CB-2 67.0 2492 95.5 423 364
668 448 456 541 CB-3 63.5 699 94.3 103 25 119 803 64 72 CB-4 Sample
leaked - not sterile - not set up CB-5 32.0 246 97.2 108 Sample had
leaked, was set up - cultures were contaminated CB-6 Not Received
CB-7 51.0 352 96.6 107 6 34 2 6 2 CB-8 37.1 197 95.6 45 10 20 6 13
14 CB-9 53.4 374 96.0 98 10 163 75 85 61 CB-10 72.4 1138 96.5 149
39 298 128 119 113 CB-11 55.6 862 93.9 71 1 23 37 34 21 CB-12 41.5
328 1.5 1 Cell viability too low - not set up CB-13 52.0 468 2.8 4
" CB-14 72.8 910 96.4 117 59 115 138 122 103 Blood had leaked CB-15
68.7 1637 91.1 126 25 76 108 86 53 .sup. " CB-16 40.2 302 92.7 28 4
8 5 8 6 .sup. " CB-17 34.1 193 93.5 26 0 0 4 9 4 CB-18 Sample
leaked - Not sterile - not set up CB-19 .sup. " CB-20 .sup. " CB-21
43.5 283 93.1 25 1 4 1 2.5 1 CB-22 55.0 1430 93.7 96 10 52 4 40 23
Blood leaked CB-23 79.8 1373 94.3 362 188 507 362 290 275 CB-24
68.0 952 92.7 72 7 33 19 39 37 CB-25 26.5 106 21.3 0.2 Cells
clumped after Ficoll Separation, assay not set up because of low
yield CB-26 40.2 181 95.3 83 12 46 42 51 32 CB-27 40.0 304 90.9 21
0 2 1 2 1 CB-28 38.7 375 93.5 7 0 0 0 0 0 CB-29 38.3 383 96.7 32 3
12 6 6 9 CB-30 29.5 139 96.7 3 0 0 0 0 0 CB-31 46.3 597 95.0 81 21
96 47 44 47 CB-32 21.4 64 94.4 4 0.2 0.6 1 1 0.2 CB-33 38.4 442
95.6 86 21 72 72 46 48 CB-33 41.0 426 97.6 98 33 90 92 71 49 CB-34
26.6 420 81.4 42 18 51 23 23 18 CB-35 35.5 579 95.7 61 28 79 39 40
27 CB-36 56.4 812 87.9 48 30 74 59 64 39 CB-37 56.0 610 96.7 109 52
135 115 129 85 CB-38 21.0 11 Cells clumped + hemolysis - not set up
CB-39 22.5 18 97.7 2 1 3 2 1 1 CB-40 56.6 849 95.6 64 31 60 33 33 9
CB-41 114.0 1493 96.0 375 15 97 157 172 52 CB-42 86.0 2649 95.6 326
0 46 124 85 19 CB-43 52.5 383 73.5 24 35 86 49 23 36 CB-44 13.0 39
51.5 1 low yield + low viability - not set up CB-45 14.4 37 52.6 1
.sup. " CB-46 27.6 419 96.7 48 14 20 33 30 19 CB-47 31.6 436 95.4
94 17 19 53 66 21 samples, through CB-48 28.7 330 65.8 6 10 37 8 8
5 a mistake, were CB-49 25.6 276 60.5 6.5 12 38 7 6 5 in transit
for 48 CB-50 85.0 994 88.5 85 59 114 51 65 41 hours before receipt
CB-51 24.5 245 92.4 51 27 73 35 37 20 CB-52 23.0 262 93.2 15 16 47
19 18 13 CB-53 50.5 1353 95.4 320 256 653 506 448 186 CB-54 67.0
791 92.2 191 38 130 72 95 50 CB-55 118.4 1219 86.2 223 143 343 107
196 85 CB-56 49.5 807 95.6 40 18 49 24 35 17 CB-57 49.4 217 92.8 43
16 32 20 17 5 CB-58 41.8 719 95.2 139 36 164 100 50 33 CB-59 34.8
602 98.1 92 55 121 50 31 11 CB-60 29.5 540 81.0 109 35 59 30 76 37
CB-61 25.8 655 90.0 68 16 30 31 43 26 CB-62 29.3 735 59.5 75 13 52
64 67 43 CB-63 26.3 686 72.4 45 3 40 18 18 6 CB-64 132 2218 96.4
464 56 213 343 306 139 CB-65 fungal contamination - not set up
CB-66 102 2029 81.3 349 153 272 237 230 126 CB-67 78.8 843 93.3 251
80 120 171 161 105 CB-68 117.5 1739 71.5 265 111 265 223 170 143
CB-69 not received CB-70 40.5 251 91.2 38 4 5 4 6 2 CB-71 40.5 713
93.3 75 16 21 37 25 15 CB-72 43.7 717 91.6 61 8 17 23 18 5 CB-73
61.2 1206 70.9 178 50 78 110 78 28 CB-74 62 1376 75.1 223 67 134
156 165 89 CB-75 60 1194 73.1 174 42 139 111 125 63 CB-76
81.5 1002 90.2 115 11 106 57 80 16 CB-77 130.0 3570 97.7 798 287
1085 527 606 255 CB-78 80.0 736 93.5 66 Cells frozen away but
colony assay not set up CB-79 69.8 845 84.5 100 " CB-80 52.2 767
88.4 32 " CB-81 127 2032 89.6 325 " CB-82 55 599 84.5 13 Cells
frozen away but colony assay not set up CB-83 85.7 1465 93.8 143 66
357 177 146 120 CB-84 45.7 334 97.4 109 65 89 50 41 30 CB-85 60.8
1660 93.5 360 209 338 518 418 187 CB-86 67.2 1579 92.6 650 182 260
351 494 234 CB-87 53.0 1420 89.8 117 58 117 94 58 42 CB-88 80.0 968
98.2 198 99 340 20 36 4 CB-89 66.5 818 96.5 340 122 231 68 61 14
CB-90 33.5 188 91.5 44 8 17 3 9 0 CB-91 87.0 1105 94.2 237 19 137
52 43 5 CB-92 51.0 398 90.0 49 31 100 50 61 46 CB-93 56.2 663 96.6
143 54 77 51 69 23 CB-94 58.2 652 88.5 130 60 94 60 78 55 CB-95
57.7 710 85.5 113 41 68 72 50 43 CB-96 36.5 150 91.9 20 4 5 12 10 6
CB-97 66.2 496 96.1 53 8 20 29 27 23 CB-98 49.0 157 95.6 23 1 4 10
10 5 CB-99 38.5 239 93.9 72 7 22 37 39 23 CB-100 72.0 1058 95.5 294
Cells frozen away but colony assays not set up CB-101 28.0 134 96.1
33 .sup. " CB-102 61.3 760 70.1 203 .sup. " CB-103 33.9 193 80.7 6
.sup. " CB-104 96.5 1911 87.2 283 .sup. " CB-105 54.0 1620 91.4 159
.sup. " CB-106 78.0 3838 92.0 204 .sup. " CB-107 64.2 1669 89.2 198
.sup. " CB-108 39.0 214 54.2 12 .sup. " CB-109 49.3 458 93.6 49
.sup. " CB-110 57.5 724 92.2 147 .sup. " CB-111 52.0 502 92.1 56 8
46 25 36 30
__________________________________________________________________________
From receipt of the samples until the cells were frozen, 16 hours
were spent in processing the cells. (This time period included cell
separation on Ficoll-Hypaque, counting the cells, setting up the
progenitor cell assays, and freezing the cells).
As shown in Table III, significant total numbers of progenitors
cells were obtained even with the overnight transit time plus 16
hour processing. Even among the samples in transit for 48 hours,
viable progenitor cells were observed. There was variability among
donors in the observed number of progenitor cells. It should be
noted that the values shown in Table III represent remaining
progenitor cells after loss of progenitors due to cell separation
procedures (see Table IV, infra).
6.3. Enrichment for Human Hematopoietic Stem and Progenitor Cells:
Cell Separating Procedures
In a preferred embodiment of the invention, whole neonatal blood
can be cryogenically preserved, and used for hematopoietic
reconstitution after thawing, in order to avoid cell losses
associated with cell separation procedures. However, it is
envisioned that cell separation procedures can be used if desired,
e.g., to minimize blood storage volumes. Thus, in the examples
sections herein, cell separation procedures are described which can
be used to enrich for neonatal hematopoietic stem cells in
collected blood. Many of the procedures relate to the enrichment of
stem cells derived from adult bone marrow or adult blood, however,
it is envisioned that the same procedures, or modifications
thereof, are equally applicable to the neonatal hematopoietic stem
cells of the present invention.
Human stem and progenitor cells are present in the non-adherent,
low density, T-lymphocyte-depleted fraction of bone marrow, spleen,
and (adult and cord) blood cells. Purification or enrichment for
the stem and progenitor cells has been carried out by
Ficoll-Hypaque density separation, adherence/non-adherence
separation, and positive selection by antibody binding.
6.3.1. Density Separations
Enrichment for human hematopoietic stem and progenitor cells has
been carried out by isolation of low density (density less than
1.077 gm/cm.sup.3)cells separated by Ficoll-Hypaque (Pharmacia Fine
Chemicals, Piscataway, NJ).
The following protocol is used for samples of bone marrow or
peripheral blood:
1. Obtain sample of bone marrow or peripheral blood.
Bone Marrow--sample should be 1-5 ml containing heparin. Place in
sterile 17.times.100 mm tube. Add 2-3 ml of sterile DPBS
(phosphate-buffered saline without magnesium or calcium). Mix
well.
Whole Blood--Dilute sample at least 1:1 with McCoy's 5A medium or
DPBS. Adjust volume to a multiple of 20 ml.
2.
Bone Marrow--spin for 10 minutes at approximately 400.times.g (1500
rpm; Beckman TJ-6R rotor) at 4.degree. C.
Whole Blood--Go to next step.
3.
Bone Marrow--Remove buffy coat and wash once with DPBS. Resuspend
to a final volume of 20-40 ml (if 1-2 pulls of 2 ml each, resuspend
to 20 ml; if 3-5 pulls, resuspend to 40 ml). Count cells; adjust
volume to a maximum of 6.times.10.sup.7 cells per 20 ml.
Whole Blood--Go to next step.
4.
Bone Marrow--With a 10 ml pipet, carefully layer 20 ml of buffy
coat suspension onto 15 ml of Ficoll-Hypaque in a 50 ml
polypropylene tube.
Whole Blood--With a 10 ml pipet, carefully layer 20 ml of blood
suspension onto 15 ml Ficoll-Hypaque in a 50 ml polypropylene
tube.
5. Using a balance, carefully adjust weight of tube(s) with
blank(s).
6. With slow acceleration, centrifuge sample(s) at 400 .times. g
(1500 rpm) for 30 minutes at 4.degree. C. Turn brake off.
7. Carefully remove the low density band and place it in a clean
sterile tube. Dilute at least 1:10 with McCoy's 5A medium or
DPBS.
8. Wash cells twice by centrifugation at 400 .times. g for 10
minutes at 4.degree. C. Resuspend to 50 ml and repeat.
9. Final resuspension should be to 10-15 ml with McCoy's media.
10. Perform cell count.
The following modification of the above procedure has been used for
cord blood separations (and was used in obtaining the data shown in
Tables III and IV):
1. Obtain cord blood, aseptically, using a 60 cc syringe containing
3000-4000 units of preservative-free sodium heparin or ACD as an
anticoagulant.
2. Perform low density cell separation using Ficoll-Hypaque density
gradient, by diluting cord blood with sterile DPBS (phosphate
buffered saline without Mg.sup.++, Ca.sup.++), pH 7.0, at a ratio
of 1:3 (cord blood: PBS). Layer 20 ml of blood suspension on 15 ml
of Ficoll-Hypaque in a 50 ml polypropylene centrifuge tube (FIG.
3). Centrifuge at 4.degree. C., 400 .times. g, for 30 minutes.
3. Collect and pool all low density cell bands (FIG. 4) from each
individual donor. Make sure that very little Ficoll-Hypaque is
collected with cells, or the cells may not pellet through the
collected Ficoll. Dilute the cell suspensions 1:1; if "X" ml of
cells were collected, dilute with "X" ml of DPBS in order to dilute
collected Ficoll sufficiently to allow cells to pellet. Pellet
cells by centrifugation at 4.degree. C., 200 .times. g, for 10
minutes.
4. Aspirate and discard supernatant from each pellet. If several
tubes were used, pool identical donor pellets after resuspending
each pellet with 5 ml of DPBS. Pellet cells by centrifugation at
4.degree. C., 200 .times. g, for 10 minutes.
5. Aspirate and discard supernatant. Resuspend pellet in 10 ml of
DPBS using a 10 ml pipet and gentle up-down aspirations. Bring
volume to 50 ml with DPBS. Pellet cells by centrifugation at
4.degree. C., 200 .times. g for 10 minutes.
6. Resuspend in RPMI-1640 medium supplemented with 5% autologous
plasma or heat-inactivated fetal calf serum (FCS). Perform cell
counts and viability. Keep cell suspension chilled to 4.degree.
C.
The effect of various density separation procedures on the yield of
progenitor cells in the human cord blood collected (described in
Section 6.1, supra) was assessed. We have compared the number of
progenitors in whole blood, no separation treatment, to that of
whole blood in which the mature erythrocytes were lysed by
treatment with ammonium chloride (NH.sub.4 Cl), low density cells
after Ficoll-Hypaque separation (density less than 1.077
gm/cm.sup.3), heavy density cells after Ficoll-Hypaque separation,
and heavy density cells after treatment with NH.sub.4 Cl to lyse
the erythrocytes (Table IV, Exp. 1).
TABLE IV
__________________________________________________________________________
COMPARISON OF HEMATOPOIETIC PROGENITOR CELLS OBTAINED WITH
DIFFERENT CELL SEPARATION PROCEDURES Progenitor Cells .times.
10.sup.-3 Separation CFU-GM CFU-GM CFU- Procedure day 7 day 14
BFU-E-2 BFU-E-1 GEMM
__________________________________________________________________________
Exp #1 None (Whole Blood) 167 220 330 356 356 Whole Blood +
NH.sub.4 Cl 55 112 43 39 43 Low Density (Ficoll) 35 87 49 23 36
Heavy Density (Ficoll) 49 104 71 82 153 Heavy Density + NH.sub.4 Cl
17 40 17 14 11 Exp #2 None (Whole Blood) 561 1020 612 484 408 Whole
Blood Sedimented 157 388 212 286 111 with Methyl Cellulose Low
Density (Ficoll) 256 653 506 448 186 Heavy Density (Ficoll) 3 8 8
14 5 Exp #3 Sample CB-57 Whole Blood Sedimented 6 12 11 12 6 with
Methyl Cellulose Low Density (Ficoll) 35 59 30 76 37 Sample CB-58
Whole Blood Sedimented 5 14 14 17 7 with Methyl Cellulose Low
Density (Ficoll) 16 30 31 43 26 Sample CB-59 Whole Blood Sedimented
6 21 31 39 17 with Methyl Cellulose Low Density (Ficoll) 13 52 64
67 43 Sample CB-60 Whole Blood Sedimented 2 9 5 4 0.4 with Methyl
Cellulose Low Density (Ficoll) 3 40 18 18 6
__________________________________________________________________________
As shown in Table IV, Exp. 1, there are many more progenitors
detected in the unseparated blood than in the low density Ficoll
preparation. This difference is not due to loss of cells into the
dense fraction of Ficoll, which contains mainly mature neutrophilic
granulocytes. Lysing whole blood erythrocytes also resulted in a
lower yield of progenitors. In experiment number 2, we compared
whole blood, whole blood that was sedimented with methyl cellulose
to remove erythrocytes, and low and high density Ficoll separated
cells. The results demonstrated that whole blood contained the most
progenitors, some of which were lost from the fraction of cells
obtained after sedimentation of the erythrocytes with methyl
cellulose. As seen in both experiments 2 and 3 of Table IV,
sedimenting cells with methyl cellulose was inferior to the low
density fraction of Ficoll with respect to numbers of progenitors.
While the Ficoll separation removed mature granulocytes and
erythrocytes from the progenitor cell fraction, some progenitors
were also lost, relative to whole blood, using this procedure.
6.3.2. Adherence/Non-Adherence Separation
An adherence/non-adherence separation protocol for enrichment of
hematopoietic stem and progenitor cells is as follows:
1. In a 60 mm Corning tissue culture dish, seed
10-15.times.10.sup.6 low density cells in up to 3 ml of McCoy's 5A
(supplemented) media with 10% fetal calf serum
(heat-inactivated).
2. Incubate for 1.5 hours at 37.degree. C. in an atmosphere of 5%
CO.sub.2.
3. Gently swirl plate to loosen non-adherent cells. Pipet into
sterile centrifuge tube. Carefully rinse dish with 3 ml McCoy's
media and pool the media.
4. Add 1 ml McCoy's media to the dish, and gently remove the cells
with a sterile rubber policeman. Remove the cells and place them in
a sterile centrifuge tube. Rinse the dish with 3 ml media and pool
media.
5. Pellet cells by centrifugation at 400 .times. g for 10 minutes
at 4.degree. C. Aspirate the supernatant and resuspend the cells in
media.
6. Repeat step 5.
7. Perform cell count.
6.4. Cryopreservation of Cord Blood Stem and Progenitor Cells
The following protocol has been used for cryopreservation of viable
hematopoietic stem and progenitor cells derived from human cord and
placental blood:
1. Pellet low density, Ficoll-separated cells by centrifugation at
4.degree. C., 200 .times. g for 10 minutes.
2. Check viable cell count by trypan blue exclusion (Kuchler, R.J.,
1977, Biochemical Methods in Cell Culture and Virology, Dowden,
Hutchinson & Ross, Stroudsburg, Pa., pp. 18-19) and manual cell
counting using a hemocytometer.
3. Gently resuspend cells to a concentration of 4.times.10.sup.6
viable cells/ml, using a mixture of cold (4.degree. C.) 50%
autologous plasma/RPMI-1640 or 50% heat-inactivated FCS/RPMI, and
place the suspension on ice.
4. In a cryovial containing 1 ml of a chilled, sterile
cryoprotective medium of 20% DMSO/RPMI-1640, carefully layer a 1 ml
portion of the aforementioned cell suspension on top of the
cryoprotective medium.
5. Approximately 10 minutes prior to freezing, slowly invert the
1:1 mixture to promote mixing, then place it on ice to allow
equilibrium between the cells and the cryoprotective medium. NOTE:
The "layered" tube should not remain unfrozen for very long, so
freezing should preferably be done within 20-30 minutes after
exposure of cells to DMSO/RPMI solution.
6. Place the vials in a freezing rack, which in turn is placed in a
4.degree. C. methanol bath, just deep enough to cover the cell
suspension (FIG. 4). This is then placed in the bottom (to ensure
proper temperature) of a -80.degree. C. freezer for at least 2
hours and less than 24 hours.
7. After cells reach the frozen state, carefully and quickly
transfer them to a long term liquid nitrogen containment vessel. A
cryogenic storage vessel which can be used is the LR1000
refrigerator (Union Carbide Corp., Indianapolis, Indiana) which
accommodates up to 40,000 cryules.
6.5. Cell Thawing
The following protocol has been used for thawing of cryopreserved
cord blood stem and progenitor cells:
1. Remove vial of frozen cells from liquid nitrogen. Immediately
thaw cell suspension by gently agitating the vial in a 37.degree.
C. water bath until just a small amount of ice remains.
2. Aseptically, begin to add drop-wise, a chilled mixture of 50%
autologous serum/RPMI-1640 medium or 50% FCS/RPMI-1640 medium with
a slight mixing between each drop, until the suspension volume is
doubled.
3. Transfer this suspension to a larger centrifuge tube (12-15 ml)
and continue to add, drop-wise, 50% serum/RPMI mixture with mixing
between every other drop until the volume reaches 6-7 ml. Diluent
may now be added, drop-wise, with mixing at every 0.5 ml increment
until the volume reaches 9-10 ml. (NOTE: The reason for stepwise
addition of diluent is to prevent osmotic shock to the cells as
DMSO is diluted in the cell suspension.)
4. Pellet cells by centrifugation at 4.degree. C., 200 .times. g,
for 10 minutes. Aspirate the supernatant.
5. Slowly add, drop-wise, 1 ml of chilled 20% autologous
serum/RPMI-1640 mixture to the pellet. "Resuspend" the pellet by
gently "flicking" the tube with a finger. After the pellet is
resuspended (clumps may remain), resuspend it further by gently
aspirating up and down with a 1 ml pipet.
6. Add an additional 4 ml chilled 20% autologous serum/RPMI,
dropwise, with mixing between every drop; then add 0.5 ml as volume
increases, as previously described.
7. Pellet cells by centrifugation at 4.degree. C., 200 .times. g,
for 10 minutes. Aspirate the supernatant.
8. Resuspend with 2-5 ml of chilled 20% serum/RPMI mixture.
9. Perform cell counts (by use of a hemocytometer) and viability
testing (by trypan blue exclusion).
Loss of cells due to clumping during the stepwise removal of DMSO
can be diminished by including DNase (20 U per 2.times.10.sup.6
cells) or low molecular weight dextran and citrate (to reduce the
pH to 6.5).
6.6. Human Hematopoietic Stem and Progenitor Cell Assays
Assays which can be used to quantitatively assess human
hematopoietic stem and progenitor cells are described in the
following examples sections. The assays for granulocytemacrophage
(CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM)
progenitor cells (Sections 6.6.1 and 6.6.2) were used to derive
part of the data for human cord blood cells that is presented in
Table III, supra.
6.6.1. CFU-GM Assay
The following assay has been used to quantify CFU-GM:
1. Obtain a suspension of cells (cord blood, bone marrow, spleen,
cell line, etc.) at a known cell concentration. The cell suspension
concentration should be at least 10 fold greater than the final
concentration desired in the plate.
2. Depending on the number of plates to be plated, the volume of
the culture mixture will vary. As an example, a 10 ml suspension
can be made, as described:
In a 17.times.10 mm polystyrene tube, combine the following
components except for the Agar (0.6%) and cells.*
______________________________________ 10 ml
______________________________________ Agar (0.6% w/v) 5 ml (50%)
(bacto-agar, Difco Corp.) **2X McCoys 5A 2 ml (20%) FCS (heat
inactivated) 1 ml (10%) ***Stimulator 1 ml (10%) *Cells 1 ml (10%)
______________________________________ *The cells are added just
before adding the melted Agar, in order to avoid allowing the cells
to sit in 2.times. McCoys for very long. Since the Agar has been
boiled, care should be taken to allow it to cool sufficiently.
**This volume may vary if the cells are more concentrated than
desired. Example: if cells were to be plated at a final cell
concen- tration of 1 .times. 10.sup.5 cells/ml, and the stock cell
suspension was 5 .times. 10.sup.5 cells/ml instead of 1 .times.
10.sup.6 cells/ml, 0.2 ml cells would be used, plus 0.8 ml of
2.times. McCoys to achieve a 1 ml volume. In general, whatever
volume is lacking after adding the other com- ponents, is made up
with 2.times. McCoy's. (See Section 6.6.1.1, infra for the
preparation of McCoy's medium). ***Colony formation can be
stimulated by factors present in med- ium conditioned by the 5637
urinary bladder carcinoma cell line (see Section 6.6.1.2 infra),
which was used routinely, or medium conditioned by the PHAL cell
line (phytohemagglutinin-stimulated leukocytes from patients with
hemochromatosis; Lu, L. and Brox- meyer, H.E., 1985, Exp. Hematol.
13:989-993) or by purified growth factors. Growth factors which may
be tested for human colony stimulation include but are not limited
to interleukin-3 (IL-3), granulocyte-macrophage (GM)-colony
stimulating factor (CSF), granulocyte (G)-CSF, macrophage (M)-CSF
(also referred to as CSF-1), erythropoietin, IL-1, IL-4 (also
called B-cell growth factor) and E-type prostaglandins. (These
molecules are available in purified form from various companies,
e.g., Cetus, Immunex, and Amgen.) For murine cell assays, pokeweed
mito- gen spleen cell conditioned media may be used (see Section
6.6.1.3, infra).
3. After the Agar has sufficiently cooled, add the appropriate
volume of cells and 0.6% Agar.
4. Place a cap on the tube, and mix the suspension well. A vortex
may be used, but with caution.
5. With an appropriate pipet, place 1 ml of the culture suspension
into a 10.times.35 mm dish, containing colony stimulating factors
if so desired. After all the dishes have been plated, allow them to
solidify.
6. Label the tray of plates and place it in the appropriate
incubator Incubation is conducted in a humidified atmosphere of 5%
CO.sub.2 at low oxygen tension (5% O.sub.2) for 7 days and 14 days.
Low oxygen tension enhances the detection of CFU-GM, BFU-E, and
CFU-GEMM cells.
7. Remove plates from the incubator and score by observation of
colonies under an inverted or stereoscopic microscope. Colonies
scored at 7 and 14 days represent maturation stages of CFU-GM
cells. (Day 7 CFU-GM represent a later or more mature progenitor of
the granulocyte-macrophage lineage, while day 14 CFU-GM represent
an earlier progenitor of the granulocyte-macrophage lineage).
6.6.1.1. Preparation of McCoy's 5A Medium
The following procedure was used to prepare 1.times. McCoy.times.s
5A Medium:
1. 1 envelope McCoy's 5A medium (Gibco #430-1500) NaHCO.sub.3, 2.2
gm. Bring to 1 liter with double-distilled H.sub.2 O; pH to
7.0-7.2.
2. Filter-sterilize by use of a 0.2 um filter and peristaltic pump
(positive pressure).
3. If medium is to be used for growth or incubation, it is
supplemented with the following:
______________________________________ Per Liter of Media
______________________________________ 8 ml MEM essential amino
acids (Gibco #320-1130) 4 ml MEM non-essential amino acids (Gibco
#32- 1140) 10 ml MEM sodium pyruvate (Gibco #320-1360) 4 ml MEM
Vitamins (Gibco #320-1120) 10 ml Penicillin-Streptomycin (Gibco
#600-5140) 15 ml Serine/Asparagine/Glutamine mixture (see recipe
infra) ______________________________________
To prepare 2.times. McCoy's 5A medium (for plating), follow the
same procedure as for 1.times., except bring the volume only to 500
ml instead of 1 liter. Add the same volume of supplements.
The Serine/Asparagine/Glutamine mixture is prepared according to
the following:
______________________________________ L-asparagine (Sigma #
A-0884) 800 mg L-serine (Sigma #S-4500) 420 mg L-glutamine (Gibco
#320-5030) 200 ml ______________________________________
1. Dissolve serine and asparagine in 450 ml double-distilled
H.sub.2 O), bring the volume to 500 ml and filter-sterilize through
a 0.2 um filter
2. Add to this sterile mixture 200 ml of L-glutamine. Mix well and
aliquot into 7.5 ml/tube. Store at -20.degree. C.
6.6.1.2. Preparation of Human 5637 Urinary Bladder Carcinoma Cell
Line Conditioned Medium
The following procedure can be used to obtain medium conditioned by
the human 5637 urinary bladder carcinoma cell line:
1. Thaw and start cells from frozen stocks, per "Quick Thaw"
protocol of Section 6.5, supra. Grow 5637 cells to confluence in a
150 cm.sup.2 flask containing 50 ml of the following medium:
RPMI 1640
glutamine (2 mM)
penicillin-streptomycin (10 ml/liter at 1000 units/ml)
10% fetal bovine serum (heat-inactivated)
2. Incubate in an atmosphere of 5% CO.sub.2, with normal O.sub.2,
for 3-5 days; check daily.
3. Split the cells 1:20 into 20.times.150 cm .sup.2 flasks with 50
ml RPMI 1640 media (as above).
4. Incubate for 7 days in 5% CO.sub.2, normal O.sub.2
5. At 3-5 days, if desired, select 1 flask of cells to prepare for
freezing as a stock supply of cells. Freeze 10.sup.6 cells/vial (1
ml) for liquid nitrogen storage, per the protocol described in
Section 6.4, supra.
6. At 7 days, collect cell medium into 20.times.50 cc centrifuge
tubes. Spin down cells and cell debris by centrifugation for 10
minutes at greater than or equal to 500 .times. g.
7. Pool the medium, filter-sterilize it using a 0.2 um filter, and
aliquot into 100 ml bottles. Store frozen at less than 0.degree. C.
(usually -20.degree. C. or -80.degree. C.).
8. Assay stimulation activity of the medium by the CFU-GM assay
described in Section 6.6.1, e.g., using human marrow cells.
6.6.1.3. Preparation of Murine Pokeweed Mitogen Spleen Cell
Conditioned Medium
The following procedure can be used to obtain murine pokeweed
mitogen spleen cell conditioned medium (PWMSCM), a crude source of
growth factors for use in hematopoietic colony stimulation for
mouse cells.
1. Obtain a single cell suspension of CBA/J mouse spleen cells at a
known cell concentration of greater than or equal to
20.times.10.sup.6 cells/ml (Mice should be 5-7 weeks old).
2. Make a large volume of cell growth suspension as follows:
______________________________________ CBA/J spleen cells 2 .times.
10.sup.6 cells/ml Heat-inactivated FCS 10% Pokeweed mitogen 0.333%
(1:300) (Gibco #670-5360) Iscove's modified Dulbecco's Remainder
media* + 3.024 gm NaHCO.sub.3 (Gibco #78-5220)
______________________________________ *Preparation described infra
in Section 6.6.2.3.
3. After mixing the above ingredients, place 50 ml of the mixture
in a 50 cm.sup.2 tissue culture flask, and incubate for 7 days at
37.degree. C. in an atmosphere of 5% CO.sub.2.
4. After seven days, collect the conditioned media and remove the
cells by centrifugation at greater than or equal to 500 .times. g
at 4.degree. C. for 10-15 minutes.
5. Carefully remove the conditioned media from the tubes, and
filter-sterilize the media by passage through a 0.45 um filter.
Store the conditioned media in 50 ml polyethylene tubes at
-20.degree. C.
6.6.2. BFU-E-2 and BFU-E-1/CFU-GEMM Assay
The following assays have been used to quantify BFU-E-2 and
BFU-E-1/CFU-GEMM. BFU-E-1 and BFU-E-2 are erythroid progenitor
cells that are operationally defined, and are not proven to be
physiologically distinct. The BFU-E-1 is operationally defined as
an early erythroid progenitor cell capable of producing a colony of
erythroid progeny cells in semi-solid medium, upon stiumlation by
erythropoietin, hemin (optional), and a burst-promoting factor. The
BFU-E-2 is operationally defined as a more mature erythroid
progenitor cell, capable of producing a colony of erythroid progeny
cells in semi-solid medium, upon stimulation by erythropoietin and
by hemin (optional). BFU-E-1 colonies tend to be larger than
BFU-E-2 colonies.
For the BFU-E/CFU-GEMM assay, Iscove's modified Dulbecco's medium
(IMDM) was used, with methyl cellulose as the semi-solid support
medium. (This was in contrast to the CFU-GM assay, where McCoy's
medium was used, with bacto-agar as the semi-solid support
medium.)
The procedure was the following:
1. Obtain a single cell suspension of known concentration of the
appropriate type of cells.
2. Depending on the number of plates plated, the volume of the
culture mixtures will vary. As an example, we will make a 3 ml
mixture. In order to increase the mixture volume, simply increase
component volumes proportionately. In a 17.times.100 mm tube, mix
the following components:
______________________________________ 3 ml ml
______________________________________ Methyl cellulose (2.1%) 1.4
ml Glutamine (200 mM, Gibco) 30 ul (2 mM) 2-mercaptoethanol
(10.sup.-2 M) 10 ul (5 .times. 10.sup.-5 M) (7 ul into 10 ml
McCoy's 5A) *Hemin (4 mM) 75 ul (0.1 mM) **Erythropoietin (20
units/ml) 0.15 ml (1 unit/ml) FCS (not heat-inactivated) 0.9 ml
(30%) Cells (at least 10 .times. desired) 0.3 ml ***Iscove's
Modified Dulbecco's 0.135 ml Medium (IMDM) ***GM Stimulator (if
desired) 0.01 ml ______________________________________
*Preparation described infra; Lu, L. and Broxmeyer, H.E., 1983,
Exp. Hematol. 11(8):721-729. **Note that there are different types
of erythropoietin which can be used as an example, Hyclone
erythropoietin has been used in murine cell assays and Toyobo
erythropoietin has been used in human cell assays. Purified
recombinant erythropoietin is commercially available (e.q., Amgen,
Thousand Oaks, CA) and may be used. ***GM stimulators include but
are not limited to various factors which ca be tested for colony
stimulation, as described for the CFUGM assay. The volume of the GM
stimulator, and thus of the IMDM, may vary with the type of
stimulator used (e.g., mouse = PWMSCM; Human = 5637 CM or PHALCM).
Als note that IMDM is strictly a compensation for the remaining
volume of 3 ml.
3. Mix suspension thoroughly by vortexing and inversion of
tubes.
4. After allowing bubbles to rise from the mixture, place 1 ml
mixture in each of two 10.times.35 mm culture plates containing
erythropoietin, hemin, and colony stimulating factors, if so
desired. Rotate the plates so that the mixture coats the surface of
the plates.
5. Place these 2 plates in a large 15.times.100 mm petri dish along
with a 10.times.35 mm humidifying dish containing about 1 ml of
H.sub.2 O. Replace the lid of the large dish.
6. Place the petri dish in an appropriate incubator for 14 days.
Conditions of incubation are the same as described for the CFU-GM
assay of Section 6.6.1.
7. Remove plates from the incubator and score by observation of
colonies under an inverted or stereoscopic microscope.
In some cultures, the GM stimulator/burst-promoting activity (e.g.,
medium conditioned by 5637 cells or PHALCM) can be omitted; under
these conditions, the assay detects a more mature population of
BFU-E (BFU-E-2) cells and few or no CFU-GEMM cells.
6.6.2.1. Preparation of 2.1% Methyl Cellulose
The 2.1% methylcellulose, for use in the BFU-E/CFU-GEMM assay, was
prepared as follows:
______________________________________ Stock solution:
______________________________________ 2.1% Methocel (Dow Chemical
Co.) 21 grams Boiling water 500 ml 2x IMDM 500 ml
______________________________________
Procedure
The gram weight of methyl cellulose is put into a sterile 3 liter
Erlenmeyer flask (having a sterile stopper) containing a sterile
magnetic flea on a large magnetic stirrer. To prevent as little
frothing as possible, stirring is initiated while 500 ml of sterile
boiling distilled H.sub.2 O is gently poured down the sides of the
flask. Stirring continues at room temperature until the flask
gradually cools (this may take an hour). When the flask is no
longer hot to the touch, 500 ml of 2.times. IMDM, which had been
allowed to come to room temperature, is added to the flask without
frothing. The flask is stoppered and transferred to the cold room
(4.degree. C.) where stirring continues for 48 hours. The solution
is then sterilely aliquoted into sterile 100 ml bottles. The
bottles are stored frozen for up to 6 months (protected from
light).
6.6.2.2. Preparation of Hemin
The hemin, for use in the BFU-E/CFU-GEMM assay, was prepared as
follows:
______________________________________ 260 mg Hemin (Eastman Kodak
#2203) 4 ml 0.5 M NaOH 5 ml Tris buffer, 1 M, pH 7.8 (approximately
9.5 parts acid to 3 parts base)
______________________________________
Bring to 100 ml with double-distilled H.sub.2 O.
1. Dissolve hemin in NaOH completely before adding Tris buffer and
H.sub.2 O.
2. After adjusting the volume to 100 ml, filter-sterilize by
passage through an 0.45 um filter, and store in 2-3 ml aliquots at
-20.degree. C.
6.6.2.3. Preparation of Iscove's Modified Dulbecco's Medium
1.times. Iscove's Modified Dulbecco's Medium (IMDM), for use in the
BFU-E/CFU-GEMM assay, was prepared as follows:
1. Measure out 5% less water (deionized, distilled) than desired
total volume of medium, using a mixing container that is as close
to the final volume as possible.
2. Add powder medium (Gibco Laboratories, Formula No. 78-220), to
water with gentle stirring at room temperature (do not heat
water).
3. Rinse out the inside of the package, to remove all traces of the
powder.
4. Add 3.024 grams of NaHCO.sub.3 per liter of medium.
5. Dilute to the desired volume with water. Stir until
dissolved.
6. Do not adjust pH. Keep container closed until medium is
filtered.
7. Sterilize immediately by Nalgene filtration.
To prepare 1 liter of 2.times. liquid medium, follow the above
procedure, except use 2 envelopes of powder instead of one, and
6.048 gm NaHCO.sub.3.
6.6.3. Stem Cell Colony Forming Unit Assay
The assay used for stem cell (S-cell) quantitation does not
directly assay self-renewal, but instead assays for the ability to
generate secondary multilineage colonies on replating. This assay
is done essentially the same as the BFU-E/CFU-GEMM assays, except
that cultures are scored after 21-28 days of incubation rather than
after 14 days (for BFU-E and CFU-GEMM). The drug
4-hydroperoxycyclo-phosphamide (4HC) appears to spare immature
progenitors at the expense of mature progenitors, and may be useful
for pretreating cells before assay. Factors which can be tested for
increasing the self-renewal ability of S-cells in vitro (thus
increasing assay efficiency) include but are not limited to hemin,
oxygen tension (Smith, S. and Broxmeyer, H.E., 1986, Brit. J.
Haematol 63:29-34), superoxide dismutase, glucose oxidase, IL-3,
GM-CSF, G-CSF, M-CSF, erythropoietin, IL-1, IL-4, etc.
6.6.4. Assay of the Proliferative Status of Stem and Progenitor
Cells
The proliferative status of stem and progenitor cells can be
measured by a high specific activity tritiated thymidine (.sup.3
HTdr) kill (or suicide) technique, carried out as follows:
1. In two small 12.times.75 mm polystyrene tubes, place the
appropriate volume of stock cell suspension containing 2-3 times
the number of cells required for plating. (For bone marrow,
2-3.times.10.sup.6 cells and for spleen, 15-20.times.10.sup.6
cells. For cord blood: 2-3.times.10.sup.6 (approx.) cells.) Label
them a and b.
2. Pellet the cells by centrifugation at 200-400 .times. g at
4.degree. C. for 10 minutes.
3. Carefully remove and discard the supernatant.
4. Add 0.5 ml of McCoy's 5Amedium supplemented as prescribed in
Section 6.6.1.1, supra, and with FCS at 10% v/v.
5. To tube b, add 50 uCi of .sup.3 HTdr (New England Nuclear,
#NET-027X Thymidine, [methyl-.sup.3 H]-20.0 Ci/mmol; 5.0 mCi/5.0 ml
H.sub.2 O). As a control, to tube a, add 50 ul of McCoy's 5A
medium.
6. Place cap back on tubes and gently vortex in order to resuspend
cells.
7. Place the tubes in a tray also containing H.sub.2 O, in an -
incubator with an atmosphere of 5% CO.sub.2, and a temperature of
37.degree. C., for 20 minutes.
8. Add 0.5 ml (2.5 mg) of ice cold (4.degree. C.) "cold"
(nonradioactive) thymidine (Sigma #T-9250) at 5 mg/ml to each tube,
and vortex lightly. Add an additional 2 ml of ice cold McCoy's 5A
medium to each tube.
9. Pellet cells by centrifugation at 200-400 .times. g at 4.degree.
C. for 10 minutes.
10 . Aspirate the supernatant into an appropriate container (one
used for radioactive disposal), and resuspend the cells with 2 ml
cold medium. Repeat step #10.
11. Aspirate the supernatant into an appropriate container.
Resuspend with McCoy's 5A containing 10% FCS to a volume where the
cell concentration is at least 10 fold greater than the plating
concentration.
12. Keep cells on ice until ready to plate.
13. Plate and carry out colony forming assays as described supra in
sections 6.6.1 through 6.6.3.
6.7. Recovery After Freeze-Thawing of Human Hematopoietic
Progenitor Cells Derived from Cord Blood
The results of progenitor cell assays after freeze-thawing were
compared to results of the same assays obtained before
freeze-thawing, in order to assess the recovery of hematopoietic
progenitor cells from human cord blood after the freeze-thawing
process. Eight cord blood samples, obtained as described in Section
6.1, supra, and separated by use of Ficoll-Hypaque, were analyzed.
The results are shown in Table V.
TABLE V
__________________________________________________________________________
RECOVERY OF CORD BLOOD HEMATOPOIETIC PROGENITOR CELLS AFTER
FREEZE-THAWING Total Number of Hematopoietic Progenitor Cells per 4
.times. 10.sup.6 Frozen Cells Pre-/Post- No. Viable CFU-GM CFU-GM
Sample Freeze-Storage Cells .times. 10.sup.-6 Day 7 Day 14 BFU-E-2
BFU-E-1 CFU-GEMM
__________________________________________________________________________
CB-1 Pre 3.7 1332 5254 3848 2146 3108 Post 2.5 284 2162 1680 1652
1205 % Survival after Thaw 67.6 21.3 41.1 43.7 77.0 38.8 CB-2 Pre
3.8 3268 6004 4028 4104 4864 Post 1.4 1902 2688 2361 1911 990 %
Survival after Thaw 36.8 58.2 44.7 58.6 46.6 20.4 CB-3 Pre 3.8 912
4408 2964 2356 2660 Post 1.6 746 2782 1702 1826 742 % Survival
after Thaw 42.1 81.8 63.1 57.4 77.5 27.9 CB-10 Pre 3.9 1014 7800
3354 3120 2964 Post 1.2 1300 3175 1526 829 794 % Survival after
Thaw 30.8 128.2 40.7 45.4 26.6 26.8 CB-14 Pre 3.8 1900 3724 4484
3952 3344 Post 1.1 1034 2672 1194 1240 892 % Survival after Thaw
28.9 54.4 71.8 26.6 31.4 26.7 CB-15 Pre 3.6 720 2160 3096 2448 1512
Post 1.7 426 2424 1170 1062 740 % Survival after Thaw 47.2 59.2
112.2 37.8 43.4 48.9 CB-16 Pre 3.7 518 1110 592 1036 740 Post 0.8
112 548 190 280 143 % Survival after Thaw 21.6 21.6 49.4 67.9 27.0
19.3 CB-17 Pre 3.7 0 0 592 1332 592 Post 0.5 190 550 170 360 210 %
Survival after Thaw 13.5 100 100 28.7 27.0 35.5 Average % 36.1 65.6
65.4 45.8 44.6 30.5 Survival after Thaw: Range( ) (13.5-67.6)
(21.3-128.2) (40.7-112.2) (28.7-67.9) (26.6-77.5) (19.3-48.9)
__________________________________________________________________________
As shown in Table V, the average % survival after freeze-thawing
was 36.1, 65.6, 65.4, 45.8, 44.6, and 30.5, respectively, for
nucleated cells, day 7 CFU-GM, day 14 CFU-GM, BFU-E-2, BFU-E-1, and
CFU-GEMM. There was a range of variability in recovery rates.
It should be noted that the data presented in Table V reflects cell
losses incurred during Ficoll-Hypaque separations and procedures
for DMSO removal, two steps which are omitted in a preferred
embodiment of the invention (NB: DMSO should be removed before
colony assays if such are desired to be carried out).
6.8. Calculations of the Reconstituting Potential of Cord Blood
The following discussion demonstrates that individual collections
of cord blood (such as described in Section 6.1) contains
sufficient hematopoietic stem and progenitor cells to repopulate
the hematopoietic system of an individual.
A survey of published reports indicates that the number of CFU-GM
infused for autologous bone marrow reconstitution in human
patients, can be relied on as an indicator of the potential for
successful hematopoietic reconstitution (Spitzer, G., et al., 1980,
Blood 55(2): 317-323; Douay et al., 1986, Exp. Hematol.
14:358-365). By standardizing published data by patient weight, and
assuming a patient weight of 150 pounds (67.5 kilograms), the
calculated number of CFU-GM needed for successful hematopoietic
reconstitution using autologous bone marrow cells ranges from
2-425.times.10.sup.4, with faster recovery noted using greater than
10.times.10.sup.4 CFU-GM.
The data presented in Table III, supra, for 81 cord blood
collections, analyzed for day 14 CFU-GM count, shows a range of
0-109.times.10.sup.4 CFU-GM per Ficoll-Hypaque-separated individual
blood collections. Seventy samples contained greater than or equal
to 2.times.10.sup.4 CFU-GM, while thirty samples contained greater
than or equal to 10.times.10.sup.4 CFU-GM. It should be emphasized
that this data is derived from Ficoll-Hypaque-separated cells
obtained by either gravity drainage from the cord or needle
aspiration from the delivered placenta. In a preferred embodiment
of the invention, where whole blood is both frozen and infused for
therapeutic use, losses due to Ficoll-Hypaque separation can be
avoided (see Table IV and Section 6.3.1 infra for data on cell
losses incurred during Ficoll-Hypaque separations). In addition, as
mentioned in Section 6.1, supra, in recent blood collections, we
have been able to obtain volumes approximately twice as large as
shown in FIG. 2 or described in Table III, by using needle
aspirations from the delivered placenta at the root of the placenta
and in the distended surface veins, in combination with cord
drainage. Furthermore, an adjustment of the collection protocol to
provide for immediate cord clamping upon delivery should result in
receipt of greater blood collection volumes (See Yao, A.C., et al.,
Oct. 25, 1969, Lancet:871-873, wherein collected neonatal blood,
obtained by drainage from the umbilical cord and from the delivered
placenta, averaged 126.6 ml volume when the umbilical cord was
clamped in less than 5 seconds after birth). Thus, although an
analysis of the data of Table III should be adjusted for expected
losses during freeze-thawing (which losses, however, should not
exceed 35%), there should be sufficient cord stem and progenitor
cells per collection sample to successfully effect hematopoietic
reconstitution.
Furthermore, the reconstituting capacity of cord blood
hematopoietic cells may be higher than that of an equal number of
bone marrow cells. Colonies derived from cord blood cells are
usually larger in size than those derived from adult bone
marrow.
6.9. In Vitro Culture Conditions for Hematopoietic Stem and
Progenitor Cells
Culture conditions for the growth in vitro of hematopoietic
progenitor cells from human cord blood have been described in
Smith, S. and Broxmeyer, H.E., 1986, British Journal of Hematology,
Vol. 63, pp. 29-34, which is incorporated by reference herein in
its entirety. Culture media was composed of the following
ingredients:
RPMI 1640 media (Gibco Laboratories, Grand Island, NY)
10.sup.-6 M hydrocortisone (Sigma, St. Louis, MO)
5 ug/ml Vitamin D.sub.3 (U.S. Biochemical Corp., Cleveland, OH)
20% fetal calf serum, heat-inactivated (Hyolone Laboratories,
Logan, UT)
2 gm/1 NaHCO.sub.3 (Fisher Scientific Co., Fair Lawn, NJ) 100 U/ml
Penicillin
100 ug/ml Streptomycin
0.25 ug/ml Fungizone
Various conditions and factors can be tested for any effect
increasing the self-renewal ability of stem cells in vitro. These
include but are not limited to oxygen tension (see Smith and
Broxmeyer, 1986, Br. J. Hematol. 63:29-34, incorporated by
reference herein), superoxide dismutase (Sigma Chemical Co., St.
Louis, Mo.), glucose oxidase (Sigma Chemical Co.), and combinations
of various colony stimulating factors, namely interleukin-3 (IL-3),
granulocyte-macrophage (GM)-colony stimulating factor (CSF),
granulocyte (G)-CSF, macrophage (M)-CSF (CSF-1), erythropoietin,
IL-1, and IL-4 (B cell growth factor).
6.10. Mouse Dissection Protocols
Mouse bone marrow and spleen are valuable sources of murine
hematopoietic stem and progenitor cells for model studies testing
new and/or improved protocols for use with the human neonatal stem
and progenitor cells of the present invention. Procedures for
dissection of mouse bone marrow and spleen are described in
Sections 6.10.1, and 6.10.2, respectively.
6.10.1. Bone Marrow Dissection
The following procedure can be used to obtain a murine bone marrow
cell suspension:
1. Sacrifice mouse as prescribed by cervical-thoracic
dislocation.
2. Inside a laboratory hood, soak the mouse with 70% ethanol (to
avoid microbial contamination), completely wetting the fur.
3. Snip through the skin, and peel the skin down to the hip by
holding the foot with either forceps that have been soaked in 70%
ethanol, or with fingers, and pulling the skin with forceps.
4. With sterile (alcohol-treated) forceps and scissors, cut away as
much muscle tissue as possible to expose the femur.
5. Remove the tibia from the femur by cutting through the knee
cartilage/joint. Discard the tibia.
6. Remove the femur from the body by placing the sharp edge of a
scissors on the anterior side of the hip joint, and pulling the
femur in the opposite direction against the scissors, so that the
scissors fits in the fold. Snip through the joint.
7. Remove the knee end of the femur first, by snipping just the end
with a scissors. Remove the hip end from the femur by the same
method.
8. With a 10 cc syringe containing 5 ml media (McCoys 5A 1.times.)
and a 27 gauge needle, place the needle in the bone cavity via the
hip end of the bone.
9. Flush the marrow from the bone by forcing media into the cavity
with the syringe, while holding the bone and syringe over a
17.times.100 mm tube.
10. After both femurs have been evacuated, break up clumps with a
10 cc syringe and a 23 gauge needle.
11. Pellet the cells by centrifugation at 400 .times. g (1500 rpm
in a Beckman TJ-6R rotor) for 10 minutes at 4.degree. C.
12. Aspirate the supernatant and discard it.
13. Resuspend the cells with 10 ml McCoys 5A media and a pipette,
and repeat steps 11 and 12.
14. Resuspend the cells with 10 ml McCoy's 5A media with a pipette,
and count the cells (with a hemocytometer).
6.10.2. Spleen Dissection
The following procedure can be used to obtain a murine spleen cell
suspension:
1. Sacrifice mouse as prescribed by cervical-thoracic
dislocation.
2. Inside a laboratory hood, soak the mouse with 70% ethanol (to
avoid microbial contamination), completely wetting the fur.
3. Place the mouse on its abdomen and snip through its left side
skin with a sterile scissors and forceps.
4. Lift the peritoneum with the forceps, and snip through to the
abdominal cavity.
5. With the spleen in view, remove it and place it in a
60.times.100 mm dish containing 5-7 ml media.
6. Place the spleen in a sterile homogenizing screen, in the dish,
and snip it into small pieces.
7. With the plunger of a 10 cc syringe, gently work the tissue
through the screen into a dish containing media.
8. Transfer the cell suspension from the dish to a tube. Rinse the
plate with 3 ml media and pool.
9. Resuspend small pieces by transferring the cell suspension from
the tube to a 10 cc syringe, and passing it through a 23 gauge
needle twice.
10. Pellet the cells by centrifugation at 400 .times. g (1500 rpm)
for 10 minutes at 4.degree. C.
11. Aspirate the supernatant and discard it.
12. Resuspend the cells with 10 ml McCoy's 5A media and a pipette,
and repeat steps 10 and 11.
13. Resuspend the cells with 10 ml McCoy's 5A media, and count the
cells (with a hemocytometer).
6.11. Hematopoietic Reconstitution of Adult Mice with Syngeneic
Fetal or Neonatal Stem cells
The experiments described in the examples sections infra
demonstrate the hematopoietic reconstitution of adult mice with
syngeneic or Tla-congenic stem cells of fetal or neonatal
blood.
A key reference and source of citations for use in animal model
studies, which describes standards for experimental irradiation, of
mice and other mammals, at the level causing 100% mortality from
hematopoietic failure, and prevention of such mortality by
hematopoietic reconstitution (with bone marrow cells), is: Balner,
H. Bone Marrow Transplantation and Other Treatment after Radiation
Injury, Martinus Nijhoff Medical Division, The Hague, 1977, which
is incorporated by reference herein.
6.11.1. Hematopoietic Reconstitution of Lethally-Irradiated Mice
with Stem Cells in Blood of the Near-Term Fetus
The examples herein described demonstrate that stem cells in blood
of the near-term fetus are able to reconstitute the hematopoietic
system of lethally-irradiated mice.
The irradiated mice were ten (B6.times.A-Tla.sup.b)F.sub.1 hybrid
males, aged seven weeks. The mice were exposed to 862.8 rads at a
radiation dose of 107.85 rad/min for 8 minutes with a .sup.137 Cs
source. This dose constitutes the LD100/30 days, i.e., the minimum
or near-minimal Lethal Dosage causing 100% mortality within a
30-day post-irradiation period. Use of the 30-day survival endpoint
is standard because hematopoietic reconstitution is deemed
sufficient by that time, and any later mortality is therefore
attributable to causes other than hematopoietic failure.
Blood was collected from five near-term (B6-Tla.sup.a
.times.A)F.sub.1 hybrid fetuses, delivered by Caesarian section
from one mother. In this experiment, near-term fetuses were used
instead of neonates in order to ensure microbial sterility. The
genetics of donor and recipient mice provides complete
histocompatibility except for a segment of chromosome 17 bearing
the Tla marker gene. All mice were maintained previously and
throughout on acidified drinking water to eradicate pseudomonas and
similar infective organisms.
As a restorative treatment, three mice each received 0.17 ml
heparinized whole fetal blood (made up to a total volume of
approximately 0.2 ml by adding M199 medium with penicillin and
streptomycin added) by intravascular injection into a peri-orbital
vein of the eye, within two hours of irradiation. The results
(Table VI) demonstrated the resultant survival of mice
reconstituted with fetal blood stem cells, in contrast to the
observed death of mice which had undergone no restorative
treatment.
TABLE VI ______________________________________ HEMATOPOIETIC
RECONSTITUTION OF LETHALLY- IRRADIATED ADULT MICE WITH STEM CELLS
IN BLOOD OF THE NEAR-TERM FETUS 30-day Sur- Group Day of Death
vival Rate* ______________________________________ (1) Treated 14
2/3** (2) Controls: no restorative 11, 12, 12, 0/7 treatment but
conditions 13, 13, 15, otherwise identical 15
______________________________________ *All 30day survivors were
normally healthy over prolonged periods of observation, displaying
the typical postirradiation graying of the coat, and would
doubtless have experienced an approximately normal lifespan, as is
typical of reconstitution with syngeneic or nearsyngeneic cell
donors. **Later typing for the Tla marker by cytotoxicity assay of
thymocytes (Schlesinger, M., et al., 1965, Nature 206:1119-1121;
Boyse, E. A., et al., 1964, Methods in Medical Research 10:39)
established repopulation by donor cells of the injected blood.
6.11.2. Hematopoietic Reconstitution of Mice with a Lesser Volume
of Near-Term Fetal Blood But not with Adult Blood
The examples herein described demonstrate that a defined volume of
near-term fetal blood contains adequate hematopoietic stem cells to
effectively reconstitute the hematopoietic system of
lethally-irradiated mice, while the same volume of adult blood will
not effect successful reconstitution.
The irradiated mice were 20 (B6 .times.A-Tla.sup.b)F.sub.1 hybrid
males aged 7 weeks, and 10 (B6.times.A-Tla.sup.b)F.sub.1 females
aged 7 weeks. The mice were exposed to 862.8 rads at a radiation
dose of 107.85 rad/min for 8 minutes with a .sup.137 Cs source
(LD100/30 days).
Blood was collected from eight near-term (B6-Tla.sup.b
.times.A)F.sub.1 hybrid fetuses, delivered by Caesarian section
from one mother. In this experiment, near-term fetuses were used
instead of neonates in order to ensure microbial sterility. The
genetics of donor and recipient mice provides complete
histocompatibility except for a segment of chromosome 17 bearing
the Tla marker gene. All mice were maintained previously and
throughout on acidified drinking water to eradicate pseudomonas and
similar infective organisms.
As a restorative treatment, 10 mice received 0.02 ml heparinized
whole fetal blood per mouse (made up to a total volume of 0.22 ml
by adding M199 medium with penicillin and streptomycin added), and
10 mice each received 0.02 ml adult whole blood identically
treated, by intravascular injection into a peri-orbital vein of the
eye, within 2 hours of irradiation. Control mice received no
restorative treatment. The results (Table VII) demonstrated that
stem cells in a defined volume of fetal blood can successfully
reconstitute the hematopoietic system, while cells in an equal
volume of adult blood cannot.
TABLE VII ______________________________________ SUCCESSFUL
HEMATOPOIETIC RECONSTITUTION WITH A DEFINED VOLUME OF NEAR-TERM
FETAL BLOOD BUT NOT WITH ADULT BLOOD 30-day Sur- Group Day of Death
vival Rate* ______________________________________ (1) Treated with
fetal blood 10, 12, 12, 5/10** 14, 14 (2) Treated with adult blood
11, 11, 12, 0/10 12, 12, 13, 14, 14, 15, 15 (2) Controls: no
restorative 9, 10, 10, 0/10 treatment but conditions 11, 11, 12,
otherwise identical 12, 12, 15, 23
______________________________________ *All 30day survivors were
normally healthy over prolonged periods of observation, displaying
the typical postirradiation graying of the coat, and would
doubtless have experienced an approximately normal lifespan, as is
typical of reconstitution with syngeneic or nearsyngeneic cell
donors. **Later typing for the Tla marker by cytotoxicity assay of
thymocytes (Schlesinger, M., et al., 1965, Nature 206:1119-1121;
Boyse, E. A., et al., 1964, Methods in Medical Research 10:39)
established repopulation by donor cells of the injected blood.
6.11.3. Hematopoietic Reconstitution With Blood of Newborn Mice in
Volumes as Low as Ten Microliters
The examples herein described demonstrate that the stem cells in a
volume of neonatal blood as low as 10 microliters can reconstitute
the hematopoietic system of lethally-irradiated mice.
The irradiated mice were 20 (B6.times.A-Tla.sup.b)F.sub.1 hybrid
males aged 8-12 weeks. The mice were exposed to 862.8 rads at a
radiation dose of 107.85 rad/min for 8 minutes with a .sup.137 Cs
source (LD100/30 days).
Blood was collected by cervical section from eighteen (B6-Tla.sup.a
.times.A)F.sub.1 hybrid neonates, less than 24 hours old. As a
restorative treatment, 5 mice received 0.04 ml heparinized whole
neonatal blood per mouse (made up to a total volume of
approximately 0.2 ml by adding M199 medium with penicillin and
streptomycin added), (Group 1); 5 mice each received 0.02 ml (Group
2); 5 mice each received 0.01 ml (Group 3); and 5 mice received no
further treatment (Group 4, radiation control). Treatment was by
intravascular injection into a peri-orbital vein of the eye.
The genetics of donor and recipient mice provides complete
histocompatibility except for a segment of chromosome 17 bearing
the Tla marker gene. All mice were maintained previously and
throughout on acidified drinking water to eradicate pseudomonas and
similar infective organisms.
The results in Table VIII show that stem cells in extremely small
neonatal blood volumes (down to 10 ul) were able to reconstitute
the hematopoietic system.
TABLE VIII ______________________________________ SUCCESSFUL
HEMATOPOIETIC RECONSTITUTION WITH NEONATAL BLOOD VOLUMES AS LOW AS
TEN MICROLITERS 30-day Sur- Group Day of Death vival Rate*
______________________________________ (1) Treated with 0.04 ml 12
4/5** neonatal blood (2) Treated with 0.02 ml 14, 18 3/5 neonatal
blood (3) Treated with 0.01 ml 12, 12, 14, 1/5 neonatal blood 14
(4) Controls: no restorative 5, 6, 9, 10, 0/5 treatment but
conditions 11 otherwise identical
______________________________________ *All 30day survivors were
normally healthy over prolonged periods of observation, displaying
the typical postirradiation graying of the coat, and would
doubtless have experienced an approximately normal lifespan, as is
typical of reconstitution with syngeneic or nearsyngeneic cell
donors. **Later typing for the Tla marker by cytotoxicity assay of
thymocytes (Schlesinger, M., et al., 1965, Nature 206:1119-1121;
Boyse, E. A., et al., 1964, Methods in Medical Research 10:39)
established repopulation by donor cells of the injected blood.
6.11.4. Hematopoietic Reconstitution with Blood of Newborn Mice in
Volumes of 10 or 15 Microliters
The examples herein described demonstrate that the stem cells in a
volume of neonatal blood as low as 10 or 15 microliters can
reconstitute the hematopoietic system of lethally-irradiated
mice.
The irradiated mice were 15 male and 5 female
(B6.times.A-Tla.sup.b)F.sub.1 hybrids aged 10-12 weeks. The mice
were exposed to 62.8 rads at a radiation dose of 107.85 rad/min for
8 minutes with a .sup.137 Cs source (LD100/30 days).
Blood was collected by cervical section from fourteen
(B6.times.A-Tla.sup.b)F.sub.1 hybrid neonates, less than 24 hours
old. As a restorative treatment, 10 mice received 0.015 ml
heparinized whole neonatal blood per mouse (made up to a total
volume of approximately 0.2 ml by adding M199 medium with
penicillin and streptomycin added), (Group 1); 5 mice each received
0.01 ml (Group 2); and the 5 female mice received no further
treatment (Group 3, radiation control). Treatment was by
intravascular injection into a peri-orbital vein of the eye. The
donor and recipient mice were genetically identical, and thus
completely histocompatible. All mice were maintained previously and
throughout on acidified drinking water to eradicate pseudomonas and
similar infective organisms.
The results shown in Table IX reveal that stem and progenitor cells
in neonatal blood volumes of 10 or 15 microliters were able to
reconstitute the hematopoietic system.
TABLE IX ______________________________________ SUCCESSFUL
HEMATOPOIETIC RECONSTITUTION WITH NEONATAL BLOOD VOLUMES OF 10 OR
15 MICROLITERS 30-day Sur- Group Day of Death vival Rate*
______________________________________ (1) Treated with 0.015 ml
12, 12, 12, 4/10 neonatal blood 13, 13, 13 (2) Treated with 0.01 ml
12, 16 3/5 neonatal blood (4) Controls: no restorative 12, 13, 14,
0/5 treatment but conditions 17, 22 ctherwise identical
______________________________________ *All 30day survivors were
normally healthy over prolonged periods of observation, displaying
the typical postirradiation graying of the coat, and would
doubtless have experienced an approximately normal lifespan, as is
typical of reconstitution with syngeneic or nearsyngeneic cell
donors.
6.12. Hematopoietic Reconstitution for Treatment of Fanconi's
Anemia
In the example herein, we describe a procedure which was carried
out to effect the hematopoietic reconstitution of a patient by
allogeneic peripheral blood stem cell infusion, for treatment of
the genetic anemia Fanconi's syndrome.
The patient was a 5 year old white male child with Fanconi's
anemia. The patient was first noted to be pancytopenic at 24 months
of age. He was subsequently confirmed to have Fanconi's anemia by
diepoxybutane-induced chromosomal breakage assay (Auerbach, A.D.,
et al., 1979, Am. J. Hum. Genet. 31(1):77-81). The patient had
undergone no interventional therapy other than Danazol
administration. He had undergone transfusions on two occasions
(once with red cells and once with platelets).
The source of neonatal blood for the hematopoietic reconstitution
was a female sibling, who was compatible with the patient for HLA
and red cell antigens. By study of the in utero sibling's
fibroblasts obtained at amniocentesis, the sibling was found to be
a four antigen match. The chromosome breakage test (Auerback, A.D.,
et al, 1979, Am. J. Hum. Genet. 31(1):77-81) demonstrated that the
female sibling did not suffer from Fanconi's anemia.
Approximately 150 ml neonatal blood was collected from the
umbilical cord and placenta of the sibling at birth, and was
diluted 1:1 in sterile pyrogen-free saline containing DMSO to a
final concentration of 10% DMSO. The blood was then shipped under
sterile conditions by overnight mail to a processing site, where it
was frozen slowly, in transplantation bags, in a time-freezing
apparatus, and was stored in liquid nitrogen.
Prior to freezing, a sample of the diluted blood was assayed to
determine hematopoietic progenitor cell counts as described in
Section 6.6, supra. The results are shown in Table X.
TABLE X ______________________________________ HEMATOPOIETIC
PROGENITOR CELLS IN DONOR NEONATAL BLOOD Before Freezing Cord Blood
Placental Blood Total ______________________________________ Total
Nucleated 1.05 .times. 10.sup.9 1.42 .times. 10.sup.8 1.2 .times.
10.sup.9 Cells Granulocyte- 2.23 .times. 10.sup.5 0.13 .times.
10.sup.5 2.46 .times. 10.sup.5 Macrophage Progenitors (CFU-GM)
Erythroid Pro- 3.72 .times. 10.sup.5 0.25 .times. 10.sup.5 3.97
.times. 10.sup.5 genitors (BFU-E) Multipotential 3.57 .times.
10.sup.4 0.28 .times. 10.sup.4 0.39 .times. 10.sup.4 Progenitors
(CFU-GEMM) ______________________________________
After a test freeze-thaw on a sample of the neonatal blood (1 month
frozen), recovery of viable hematopoietic progenitor cells was as
follows (as assessed by in vitro hematopoietic progenitor cell
colony assays as described in Section 6.6): 100% of CFU-GM, 45% of
BFU-E, and 75% of CFU-GEMM.
The patient was conditioned for hematopoietic reconstitution by
methods similar to those which have been used for conditioning
nonconstitutional aplastic anemia (Gluckman, E., et al., 1984, in
Aplastic Anaemia, Stem Cell Biology and Advances in Treatment,
Young, N.S., et al., eds., Alan R. Liss, Inc., New York, pp.
325-333; incorporated by reference herein) except that dosages of
chemoradiotherapy were decreased. The patient was administered
cytoxin.RTM. (cyclophosphamide) intravenously at a dosage of 5
mg/kg/day at six, five, four, and three days prior to neonatal
blood infusion, for a total of 20 mg/kg. One day prior to infusion,
the patient was subjected to thoraco-abdominal irradiation with 500
rads and administered Cyclosporin A.
The frozen blood sample was shipped, under liquid nitrogen, to the
site of patient treatment, where it was thawed in a water bath.
Approximately 300 ml of the thawed blood sample was infused
intravenously into the patient for treatment of the Fanconi's
anemia.
6.13. Flowchart: Description of a Service
In a particular embodiment of the invention, the isolation and
preservation of neonatal hematopoietic stem and progenitor cells is
envisioned as a service offered to each prospective cell donor,
which can comprise the steps listed below. The description is meant
for illustrative purposes only, in no way limiting the scope of the
invention.
1. Contact
Initial contact is made between an expectant mother (client) and
the obstetrician, who arranges the service.
2. Blood Collection
In the obstetrical ward, after the infant has been delivered and
separated from the cord in the usual way, blood is drawn from the
cord into a specially designed receptacle, which is sealed and
placed in a customized shipping container, together with a
data-form, completed by a member of the obstetrical team, giving
details of the birth.
3. Transport
Once daily, an overnight freight carrier collects the shipping
containers from the obstetrical wards, and transports them to
processing headquarters by 10:30 A.M. the following day.
4. Registration
Upon receipt at headquarters, each container is catalogued. The
blood enters the laboratory for processing (optional).
5. Blood Processing (optional)
The cells are separated, and the white cells, which include the
stem and progenitor cells, are retained for storage.
6. Testing
The separated cells undergo routine testing (see Section 5.1.2,
supra). In exceptional cases, special testing may be indicated to
determine whether the sample is contaminated, e.g., by maternal
blood. Samples may be rejected for reason of contamination or other
causes.
7. Packaging and Labeling
Cells from each accepted sample are dispensed into standard
freezing vials (cryules) and labeled in conventional and
computer-generated characters.
The cells of each individual are allocated to four cryules, two of
which are assigned for storage to one freezer and two to another,
independently-serviced, freezer. A fifth cryule contains cells set
aside for testing of identity, viability, and function, when
withdrawal of cells is required for therapy.
Labels are printed by computer, using a special printer, on silk,
which withstands immersion in liquid nitrogen. The label data
include the registration number, in machine readable and human
readable characters, date of freezing, cryule number (1-4, 5) and
freezer assignment (A and B).
8. Freezing and Storage
The cryules are subjected to slow freezing, and assigned to two
separately maintained liquid nitrogen refrigerators.
9. Permanent Records
The entire preparative history is entered into the permanent
records, including location within cryostorage modules. For
example, data input for each donor for maintenance in the computer
records can comprise:
Registration number
Name
Sex
Date of birth
Place of birth (hospital identification)
Birth certificate number
Name of mother
Date of receipt of cells
Date of freezing
Freezer positions
Obstetrical data
(a) special circumstances of birth
(b) if twin, registration number of co-twin
(c) any health disorder of the mother
Test results
(a) differential cell counts
(b) bacterial cultures
(c) other
10. Notification to Client
The client is notified of the registration number, for preservation
with child's documents, and is asked for information not available
at the time of birth (given name, birth number), for inclusion in
permanent records.
11. Withdrawal of Cells for Clinical Use
Requests for cells for treatment of the donor are made on behalf of
the donor by a suitably accredited physician affiliated with an
appropriate hospital unit. Cells are withdrawn from the cell bank
and matched for identity with the recipient. The cells are also
tested for viability and microbial contamination, and quantified in
terms of stem cell, progenitor cell, and other categories. Further
tests are conducted as required. Cells and an accompanying report
are delivered to the medical institution designated by the
physician. An appropriate notation is entered in the permanent
records.
It is apparent that many modifications and variations of this
invention as hereinabove set forth may be made without departing
from the spirit and scope thereof. The specific embodiments
described are given by way of example only and the invention is
limited only by the terms of the appended claims.
* * * * *