By
From the Department of Genetics, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M551A8
Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro
progenitor assays, knowledge of the conditions that permit maintenance of primitive human
repopulating cells is lacking. We report that primitive human cells capable of repopulating
nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38
cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold
increase in the number of CD34+CD38
cells and colony-forming cells, respectively, as well as
a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were
lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture
conditions that support primitive cells.
Ex vivo culture is a crucial component of several clinical
applications currently in development including gene
therapy, tumor cell purging, and stem/progenitor cell expansion (1, 2). Ideally, each of these therapies requires
maintenance or expansion of repopulating cells without induction of differentiation. Clinical transplantation studies
have demonstrated that human hematopoietic cells can be
cultured ex vivo with or without stroma and still retain the
capacity to engraft human recipients, although it is not
known if their number is reduced (3). Moreover, all of these studies involve autologous transplantation, making it
difficult to determine whether long-term repopulation was
derived from cultured cells or from surviving endogenous
stem cells. Although clinical gene marking studies could
address the question of stem cell maintenance in an autologous setting, conclusive evidence that engraftment was derived from a pluripotent stem cell is still lacking (6, 7). It
appears that the ex vivo culture conditions for gene transfer
may not induce the cycling of repopulating cells (as required for retrovirus-mediated gene transfer; reference 8), or they may induce stem cells to differentiate resulting in
the loss of their repopulating capacity (2).
Typically, ex vivo cultures are initiated from mononuclear cells or purified CD34+ cells (1), although in some
cases, highly purified CD34+CD38 Recently, we have identified a novel human hematopoietic cell, termed the SCID repopulating cell (SRC), that is
capable of extensive proliferation and multilineage repopulation of the bone marrow of nonobese diabetic (NOD)/
SCID mice. Using cell purification and retroviral gene marking, the SRC were found to be biologically distinct from
CFC and most LTC-IC (16, 17). The SRC were found exclusively in the CD34+CD38 Cell Purification.
Samples of cord blood (CB) were obtained
from placental and umbilical tissues and the mononuclear cells
were collected on Ficoll (Pharmacia, Uppsala, Sweden). CD34+
CD38 Clonogenic Progenitor Assays.
Human clonogenic progenitors
were assayed under standard conditions (16), except that 10%
5637-conditioned medium was included.
Liquid Suspension Cultures.
CD34+CD38 Transplantation of Purified Cells into NOD/SCID Mice.
Cells were
transplanted into NOD/LtSz-scid/scid (NOD/SCID) mice according to our standard protocol (16). In all cases, cells were
cotransplanted with nonrepopulating CD34 Limiting Dilution Analysis.
High molecular weight DNA was
isolated from the BM of transplanted mice and the number of
human cells was quantified by probing with a human chromosome 17-specific Flow Cytometric Analysis of Engrafted NOD/SCID Mice.
BM of
engrafted mice was analyzed by flow cytometric analysis using the
FACScan® as described previously (17, 20). The antibody combinations used were CD14 and CD20 conjugated to FITC, CD33
and CD19, conjugated to PE, and CD45 was conjugated to peridinin chlorophyll protein. For each mouse analyzed, an aliquot of
cells was also stained with mouse IgG conjugated to FITC, PE,
and peridinin chlorophyll as isotype controls.
The proliferative capacity of CD34+CD38
To determine the maintenance of the CD34+CD38
To determine the effect of in vitro culture on the clonogenic progenitors present in the CD34+CD38 and CD34+Thy-1+
cells have been used (9). Whether primitive cells are actually expanded during ex vivo culture is controversial because different assays and culture conditions have been used
in different studies (10, 12). It has recently been demonstrated that highly purified subfractions of CD34+ cells
possess the greatest proliferative potential, resulting in a
large expansion of colony-forming cells (CFC), while long-term culture initiating cells (LTC-IC) show either a slight
reduction (15) or moderate increase (13). Although these in
vitro assays provide an essential quantitative assessment of
the functional properties of the expanded cells, they do not
evaluate repopulation capacity.
cell fraction and, under
our conditions, were poorly transduced with retroviral vectors relative to CFC and LTC-IC. A quantitative assay for
SRC was developed using limiting dilution analysis (18), enabling us to determine that there is 1 SRC in 617 CD34+CD38
cells (17). Using this quantitative assay, we
have compared the effect of ex vivo culture on SRC, CFC,
mononuclear cells, as well as CD34+CD38
and CD34+
CD38+ cell content.
and CD34+CD38+ cells were collected using our standard protocol (17). In brief, CB cells were first enriched for
CD34+ cells by negative selection using the StemSep device
(Stem Cell Technologies Inc., Vancouver, Canada). These cell
fractions were further purified on a FACStar Plus® (Becton Dickinson, San Jose, CA), based on CD34 and CD38 expression using
similar sorting gates shown previously (17).
and CD34+CD38+
cells were incubated in 50 µl of IMDM supplemented with 1%
BSA (Stem Cell Technologies Inc.), 5 µg/ml of human insulin
(Humulin R, Lilly, Canada), 100 µg/ml of human transferrin
(GIBCO BRL, Burlington, Canada), 10 µg/ml of low density lipoproteins (Sigma Chemical Co., St. Louis), 10
4 M
-mercaptoethanol and growth factors (GF). The GF cocktail was used at
final concentrations of 300 ng/ml of stem cell factor (Amgen Biologicals, Thousand Oaks, CA) and Flt-3 (Immunex, Seattle, WA),
50 ng/ml of granulocytic CSF (Amgen Biologicals), and 10 ng/
ml of IL-3 (Amgen Biologicals) and IL-6 (Amgen Biologicals). Cells were cultured in flat-bottomed suspension wells of 96-well plates (Nunc, Burlington, Canada), incubated for 4 and 9 d at 37°C and 5% CO2, and 50 µl of fresh GF cocktail was added to
each well every other day.
Lin+ cells as accessory cells. The transplanted mice received alternate day intraperitoneal injections of human cytokines (human stem cell factor, 10 µg, human IL-3 and human GM-CSF, 6 µg; provided by I. McNiece, Amgen Biologicals). Mice were killed 8-10 wk after transplantation, and bone marrow (BM) cells were collected from femurs, tibiae, and iliac crests.
-satellite probe as previously described (19). The
frequency of SRC was determined by limiting dilution analysis as
described previously (17, 18). In brief, a transplanted mouse was
scored as positive (engrafted) if any human cells were detectable in
the murine BM by Southern blot analysis. The data from several
limiting dilution experiments were pooled and analyzed by applying
Poisson statistics to the single-hit model. The frequency of SRC
was calculated using the maximum likelihood estimator (17, 18).
Measurement of the Proliferation and Differentiation of Ex
Vivo Cultures Initiated with Human CD34+CD38 and CD34+
CD38+ Cells.
and CD34+CD38+ cells were determined by comparing
the number of cells obtained after 4 and 9 d of culture to
those originally seeded (Fig.1). The purified cells were incubated in serum-free media with the addition of growth
factors based on a cocktail previously shown to expand LTC-IC 30-50-fold in 10-21 d from an initial inoculum
of CD34+CD38
cells (13). The number of cells in the
CD34+CD38
wells increased by an average of 4-fold,
whereas the CD34+CD38+ wells increased by more than
16-fold by day 4 (Fig. 1). By day 9, the CD34+CD38
wells increased by 8-fold, whereas the CD34+CD38+ wells
had expanded by 22-fold compared to day 0.
Fig. 1.
Increase in cell number after in vitro culture of CD34+
CD38 and CD34+CD38+ cells. Purified cells were counted and seeded
(25-2,000) in wells containing serum free media (day 0). Cells were harvested from individual wells after 4 and 9 d, counted, and the mean fold
increase in absolute cell number of CD34+CD38
(solid bar) and
CD34+CD38+ (shaded bar) cells was calculated.
[View Larger Version of this Image (18K GIF file)]
and CD34+CD38+ phenotypes after 4 and 9 d of culture,
individual wells were analyzed by flow cytometry (Fig. 2).
The vast majority of cells obtained after 4 d of culture in
the CD34+CD38
wells maintained the same phenotype.
However, by day 9, no CD34+CD38
cells remained and
all of the cells had begun to differentiate, as evidenced by
the large number of CD34+CD38+ cells. A similar pattern
was seen in CD34+CD38+ wells; at day 4 most of the cells
retained the same phenotype, whereas at day 9 extensive
differentiation had occurred with the majority of cells losing both CD34 and CD38 expression.
Fig. 2.
Analysis of CD34 and CD38 expression of highly purified populations before and after in vitro culture. A representative experiment (n = 3)
of CD34 and CD38 cell surface expression performed on initially purified CD34+CD38 and CD34+CD38+ cells, and purified cells after 4 and 9 d of
culture in serum-free conditions. The entire contents of individual wells was collected at 4 and 9 d (5,000-9,000 cells), stained with monoclonal antibodies, and analyzed using flow cytometric analysis.
[View Larger Version of this Image (27K GIF file)]
and CD34+
CD38+ cell fractions, plating assays were performed at day
0, 4, and 9 (Table 1). Initially, both the CD34+CD38
and
CD34+CD38+ cells had a high plating efficiency (PEf ); 1 in 3.3 and 1 in 3.4 cells gave rise to CFC, respectively. After
4 d of culture, the PE of the CD34+CD38
wells increased
to 1 in 1.1, whereas the CD34+CD38+ had decreased
slightly to 1 in 4.6. After 9 d, the PE of the CD34+CD38
wells had decreased to 1 in 4.5 cells, whereas the CD34+
CD38+ wells had decreased even further to 1 in 12.2. Overall,
much higher numbers of progenitors were recovered from
CD34+CD38+ wells than the CD34+CD38
wells. Moreover, the total number of clonogenic progenitors continued to increase between 4 and 9 d CD34+CD38+ wells,
whereas the fold increase was less in the CD34+CD38
wells by day 9.
We
have demonstrated through limiting dilution analysis that
SRC are found exclusively in the CD34+CD38 fraction
at a frequency of 1 in 617 cells (17). In the present study,
we used the quantitative SRC assay to measure the number of SRC present in the wells at days 0, 4, and 9 of ex vivo
culture. At day 0, wells were seeded with 500-1,000
CD34+CD38
cells. The contents of each well at day 4 and
9 were either transplanted into one NOD/SCID mouse or
divided into three or eight equally sized aliquots before injection into NOD/SCID mice. Human cell engraftment
was determined 6-8 wk later to determine whether an SRC
had been present in the well. The results of one representative experiment are shown in Fig. 3 A. In this experiment,
five wells were initially seeded with 500 CD34+CD38
cells. The total cell number increased fourfold to ~2,200
cells/well after 4 d in culture. The mouse transplanted with
the entire contents of one well was engrafted with human
cells. Surprisingly, two out of three mice transplanted with
730 expanded cells and two out of eight mice transplanted
with only 275 expanded cells were also engrafted (Fig. 3
A). None of the mice transplanted with cells that had been
expanded for 9 d were engrafted, despite the injection of
16-fold more cells compared to the day-4 expanded cultures (4,500 versus 275, respectively). Fig. 3 B summarizes the level of engraftment in 81 NOD/SCID mice transplanted with expanded CD34+CD38
cells, at different
doses, from 10 CB samples cultured for 4 d. The frequency
of SRC in the ex vivo-cultured cell populations was calculated from this data to be 1 SRC in 668 expanded cells (range 1 in 427 to 1 in 1,043). This frequency is similar to
that in CD34+CD38
cells before expansion (1 in 617; reference 17). Since the absolute number of cells increased
fourfold after 4-d, and there was no significant change in
the frequency of SRC, these results indicate a fourfold increase in the absolute number of repopulating cells.
Expanded SRC Give Rise to Multilineage Differentiation.
To determine whether expanded SRC possessed the same
in vivo proliferative and differentiative capacity as uncultured SRC, BM samples of engrafted mice were analyzed
by multiparameter flow cytometry. A representative analysis of an NOD/SCID mouse transplanted with 1,200 cells
expanded from 400 CD34+CD38 cells is shown (Fig. 4).
The BM of this mouse contained 0.4% human cells as detected by expression of CD45, a human-specific panleukocyte marker (Fig. 4 A). Human CD45+ cells were gated
(region R1 in Fig. 4 A) and these cells were further analyzed for their expression of lineage-specific antigens. The
isotype control is shown in Fig. 4 B. B lymphoid cells were present in the murine BM as shown by staining for CD19
and CD20 (Fig. 4, C and D). Fig. 4, E and F demonstrate
the presence of myeloid cells (CD33+) and mature monocytes (CD14+). This engraftment pattern of mice transplanted with expanded CD34+CD38
cells is similar to
that observed after transplantation of unsorted CB cells (20)
and of purified CD34+CD38
cells (17) demonstrating that
in vitro cultured SRC still possess extensive proliferative
and differentiative capacity.
The recently developed SRC assay provides a method to
identify and characterize human repopulating cells and the
factors that govern their developmental program (21). This
report provides the first demonstration that primitive human repopulating cells can be maintained and even modestly expanded during ex vivo serum-free culture using a
cocktail of cytokines. By quantitative analysis, we found
that the number of SRC increased fourfold after 4 d in culture concomitant with increases in the total cellularity, and in the numbers of CFC and CD34+CD38 cells. Despite
even greater increases in the total number of cells, CD34+
cells, and CFC, no SRC were detected when cells were
cultured for 9 d. Others have shown continued increases in
the number of LTC-IC for up to 21 d of culture (13). This
dissociation between SRC and in vitro progenitors is consistent with our earlier results from gene marking and cell
purification experiments indicating that SRC are distinct
from and more primitive than CFC and most LTC-IC assayed in 5-wk stromal cultures (16, 17). Thus, the SRC assay will play an important role in identifying factors that cause SRC to proliferate without inducing differentiation
and loss of repopulating activity and in the design of methods to transduce primitive human cells for stem cell gene
therapy.
All prior studies aimed at developing clinical applications
have used surrogate assays to optimize the conditions for ex
vivo cultures including quantification of the number of
CD34+ cells, CFC, or LTC-IC (12, 13, 22). Most conditions result in marked expansion of CFC and CD34+ cells
(1, 12, 23), with smaller increases in the number of more
primitive cells such as LTC-IC and extended LTC-IC (13,
24). Some studies have shown a decline in LTC-IC during
10 d of culture, probably because assay methods or culture
conditions differ (15). Our results indicate that total cellularity, CFC content, and the number of CD34+ cells do
not correlate with the repopulating potential of cultured cells. Moreover, the more mature CD34+CD38+ population, which has been previously shown to be devoid of repopulating cells (17), had a higher proliferative capacity
with respect to both total cellularity and CFC number
compared to cultures initiated with CD34+CD38 cells after 4 and 9 d of in vitro culture. Although we have shown that only a fraction of CD34+CD38
cells have SRC activity, this combination of cell-surface markers seemed to
provide good correlation with repopulating potential. The
number of CD34+CD38
cells increased by four- to fivefold during the first 4 d of culture, whereas all the cells had
differentiated by 9 d, consistent with the loss of SRC. We
conclude that the SRC assay will play an important role in
the development of clinical methods for ex vivo expansion.
Address correspondence to Dr. John E. Dick, Department of Genetics, Research Institute, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada, M5G 1X8. Phone: 416-813-6354; FAX: 416-813-4931; E-mail: dick{at}sickkids.on.ca
Received for publication 1 May 1997 and in revised form 13 June 1997.
M. Bhatia and D. Bonnet contributed equally to this work.We thank I. McNiece at Amgen Biologicals for cytokines, L. McWhirter for providing cord blood specimens, and members of the laboratory for critically reviewing the manuscript.
Supported by grants from the Medical Research Council of Canada (J.E. Dick), the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Canadian Genetic Diseases Network of the National Centers of Excellence (J.E. Dick), a Research Scientist award from the National Cancer Institute of Canada (J.E. Dick), a Medical Research Council of Canada Scientist award (J.E. Dick), postdoctoral fellowships from the National Cancer Institute of Canada (M. Bhatia), the Leukemia Research Fund of Canada and the Medical Research Council of Canada (J.C.Y. Wang), the Deutsche Krebshilfe (U. Kapp), and the Human Frontier Science Organization Program and the French Cancer Research Association (D. Bonnet).
1. | Moore, M.A.. 1995. Expansion of myeloid stem cells in culture. Semin. Hematol. 32: 183-200 [Medline]. |
2. | Williams, D.A.. 1993. Ex vivo expansion of hematopoietic stem and progenitor cells-robbing Peter to pay Paul? Blood. 81: 3169-3172 [Medline]. |
3. |
Brugger, W.,
S. Heimfeld,
R.J. Berenson,
R. Mertelsmann, and
L. Kanz.
1995.
Reconstitution of hematopoiesis after high-dose chemotherapy by autologous progenitor cells generated
ex vivo.
N. Engl. J. Med.
333:
283-287
|
4. | Chang, J., L. Coutinho, G. Morgenstern, J.H. Scarffe, D. Deakin, C. Harrison, N.G. Testa, and T.M. Dexter. 1986. Reconstitution of haemopoietic system with autologous marrow taken during relapse of acute myeloblastic leukaemia and grown in long-term culture. Lancet. 1: 294-295 [Medline]. |
5. |
Barnett, M.J.,
C.J. Eaves,
G.L. Phillips,
R.D. Gascoyne,
D.E. Hogge,
D.E. Horsman,
R.K. Humphries,
H.G. Klingemann,
P.M. Lansdorp, and
S.H. Nantel.
1994.
Autografting with
cultured marrow in chronic myeloid leukemia: results of a pilot study.
Blood.
84:
724-732
|
6. |
Brenner, M.K..
1996.
Gene transfer to hematopoietic cells.
N.
Engl. J. Med.
335:
337-339
|
7. | Kohn, D.B., K.I. Weinberg, J.A. Nolta, L.N. Heiss, C. Lenarsky, G.M. Crooks, M.E. Hanley, G. Annett, J.S. Brooks, A. el-Khoureiy, et al . 1995. Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nat. Med. 1: 1017-1023 [Medline]. |
8. | Mulligan, R.C.. 1993. The basic science of gene therapy. Science (Wash. DC). 260: 926-932 [Medline]. |
9. |
Petzer, A.L.,
P.W. Zandstra,
J.M. Piret, and
C.J. Eaves.
1996.
Differential cytokine effects on primitive (CD34+CD38![]() |
10. |
Hao, Q.,
F.T. Thiemann,
D. Peterson,
E.M. Smogorzewska, and
G.M. Crooks.
1996.
Extended long-term culture reveals
a highly quiescent and primitive human hematopoietic progenitor population.
Blood.
88:
3306-3313
|
11. | Lansdorp, P.M., and W. Dragowska. 1992. Long-term erythropoiesis from constant numbers of CD34+ cells in serum-free cultures initiated with highly purified progenitor cells from human bone marrow. J. Exp. Med. 175: 1501-1509 [Abstract]. |
12. |
Kobayashi, M.,
J.H. Laver,
T. Kato,
H. Miyazaki, and
M. Ogawa.
1996.
Thrombopoietin supports proliferation of human primitive hematopoietic cells in synergy with steel factor
and/or interleukin-3.
Blood.
88:
429-436
|
13. |
Petzer, A.L.,
D.E. Hogge,
P.M. Landsdorp,
D.S. Reid, and
C.J. Eaves.
1996.
Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their
expansion in defined medium.
Proc. Natl. Acad. Sci. USA.
93:
1470-1474
|
14. |
Young, J.C.,
A. Varma,
D. DiGiusto, and
M.P. Backer.
1996.
Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture.
Blood.
87:
545-556
|
15. |
Traycoff, C.M.,
S.T. Kosak,
S. Grigsby, and
E.F. Srour.
1995.
Evaluation of ex vivo expansion potential of cord
blood and bone marrow hematopoietic progenitor cells using
cell tracking and limiting dilution analysis.
Blood.
85:
2059-2068
|
16. | Larochelle, A., J. Vormoor, H. Hanenberg, J.C.Y. Wang, M. Bhatia, T. Lapidot, T. Moritz, B. Murdoch, L.X. Xiao, I. Kato, et al . 1996. Identification of primitive hematopoietic cells capable of repopulating NOD/SCID mice: implications for gene therapy. Nat. Med. 2: 1329-1337 [Medline]. |
17. |
Bhatia, M.,
J.C.Y. Wang,
U. Kapp,
D. Bonnet, and
J.E. Dick.
1997.
Purification of primitive human hematopoietic
cells capable of repopulating immunodeficient mice.
Proc. Natl.
Acad. Sci. USA.
94:
5320-5325
|
18. |
Wang, J.C.Y.,
M. Doedens, and
J.E. Dick.
1997.
Primitive
human hematopoietic cells are enriched in cord blood compared to adult bone marrow or mobilized peripherial blood as
measured by the quantitative in vivo SCID-repopulating cell
(SRC) assay.
Blood.
89:
3919-3925
|
19. | Lapidot, T., F. Pflumio, M. Doedens, B. Murdoch, D.E. Williams, and J.E. Dick. 1992. Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in scid mice. Science (Wash. DC). 255: 1137-1141 [Medline]. |
20. |
Vormoor, J.,
T. Lapidot,
F. Pflumio,
G. Risdon,
B. Patterson,
H.E. Broxmeyer, and
J.E. Dick.
1994.
Immature human
cord blood progenitors engraft and proliferate to high levels
in immune-deficient SCID mice.
Blood.
83:
2489-2497
|
21. | Dick, J.E.. 1996. Normal and leukemic human stem cells assayed in SCID mice. Semin. Immunol. 8: 197-206 [Medline]. |
22. |
Hao, Q.L.,
A.J. Shah,
F.T. Thiemann,
E.M. Smogorzewska, and
G.M. Crooks.
1995.
A functional comparison of CD34+
CD38![]() |
23. |
Bernstein, I.D.,
R.G. Andrews, and
K.M. Zsebo.
1991.
Recombinant human stem cell factor enhances the formation of
colonies by CD34+lin![]() ![]() |
24. |
Shah, A.J.,
E.M. Smogorzewska,
C. Hannum, and
G.M. Crooks.
1996.
Flt3 ligand induces proliferation of quiescent
human bone marrow CD34+CD38![]() |