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HEMATOPOIESIS
From Hematology-Oncology Section, University of
Illinois College of Medicine, Chicago, IL; Navy-NIDDK Transplantation
and Autoimmunity Branch, Bethesda, MD.
The marrow repopulating potential (MRP) of different sources of
human hematopoietic stem cells (HSCs) was directly compared using an in
vivo assay in which severe combined immunodeficient disease (SCID) mice
were implanted with human fetal bones. HSCs from 2 human lymphocyte
antigen (HLA)-mismatched donors were injected individually or
simultaneously into the fetal bones of a 3rd distinct HLA type and
donor and recipient myeloid and lymphoid cells were identified after 8 to 10 weeks. The study compared the MRP of umbilical cord blood (CB)
and adult bone marrow (ABM) CD34+ cells as well as grafts
of each type expanded ex vivo. Equal numbers of CB and ABM
CD34+ cells injected individually demonstrated similar
abilities to establish multilineage hematopoiesis. However, when CB and
ABM cells were transplanted simultaneously, the engraftment of CB cells
was markedly superior to ABM. CB and ABM CD34+ cells were
expanded ex vivo using either a porcine microvascular endothelial cell
(PMVEC)-based coculture system or a stroma-free expansion system.
Primary CB CD34+ cells or CD34+ cells expanded
in either culture system demonstrated a similar ability to engraft.
However, the MRP of expanded grafts simultaneously injected with
primary CB cells was uniformly inferior to primary CB cells.
CD34+ cell grafts expanded in the stroma-free system,
furthermore, outcompeted CD34+ cells expanded using the
PMVEC coculture system. The triple HLA-mismatched SCID-hu model
represents a novel in vivo stem cell assay system that permits the
direct demonstration of the functional consequences of ex vivo HSC
expansion and ontogeny-related differences in HSCs.
(Blood. 2000;96:3414-3421) Human hematopoietic stem cells (HSCs) represent
rare cell populations that are characterized by their unique ability to
self-renew, differentiate into multiple lineages, and rescue
myeloablated hosts.1-3 Important differences in the
functional properties of different tissue sources of HSCs have been
well documented.2,3 Some of these functional differences
appear to be based on intrinsic, ontogeny-related
properties.4-7 In addition, stem cells that have been
exposed ex vivo to cytokines possess an impaired ability to repopulate
myeloablated hosts.8-12 These acquired defects have been
attributed to a cytokine-induced reduction in stem cell self-renewal (replicative senescence) or an acquired, cell cycle-specific defect in
homing to the marrow.8,13-15
Many of the functional properties characteristic of the various tissue
sources of human HSCs are readily apparent with their use as grafts
during clinical transplantation.16-23 Cord blood (CB)
grafts are associated with delayed times to hematologic reconstitution when compared to marrow grafts, whereas mobilized stem cell grafts are
associated with shortened periods required for hematologic reconstitution.16,17,20-23 These functional properties
have been further characterized using a variety of in vitro and in vivo stem cell assays.24-33 These assays include in vitro
long-term stem cell cultures and a variety of in vivo assays in which
human stem cells engraft and differentiate in severe combined
immunodeficient disease (SCID) mice or early gestation sheep fetuses,
producing xenogenic chimerism that persists for months to
years.24-33 A direct comparison of the functional
properties of these various sources of human stem cell populations
has not, however, previously been possible.
Studies of the long-term functional capacity of various sources of
murine stem cells, however, have been achieved with the use of
competitive repopulation assays.34-40 Stem cell function of a donor with a particular genotype has been assayed by mixing its
marrow with a constant number of marrow cells from a second donor with
a distinguishable phenotypic marker and measuring the relative ability
of each donor cell population to reconstitute stem cell-depleted
recipients.34 Such competitive repopulation assays have
been useful for the study of the behavior of various sources of murine
HSCs and their behavior after ex vivo expansion.34-40 In
this report, we describe a new in vivo assay system that now permits
the direct assessment of the relative marrow repopulating capacity and
differentiative capacity of various sources of human HSCs.
Cell collection and separation
Isolation of cord blood and adult bone marrow
CD34+ cells
Ex vivo expansion cultures of cord blood and adult bone marrow CD34+ cells The porcine microvascular endothelial cell line (PMVEC) is a primary cell line derived from 4- to 6-month-old Yucatan minipig brains (Sus scrofa).41 The ability of PMVECs to support the proliferation of early human marrow cells has been previously reported by our group.11,41-43 PMVEC (passages 22 through 29) were maintained and used for hematopoietic cell expansions as previously described.11,41-43 Triplicate cultures of CB or ABM CD34+ cells were seeded into 6-well tissue culture plates (Corning-Costar, Cambridge, MA) at 3 to 15 × 104 cells/well with pre-established PMVEC monolayers (PMVEC cocultures) or into plates without PMVEC monolayers (stroma cell-free cultures). All cultures were maintained in IMDM with 10% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin, 1 mg/mL streptomycin (all from BioWhittaker) in a humidified incubator maintained at 37°C with 5% CO2. Cultures were supplemented with a combination of recombinant human cytokines including interleukin (IL)-3 at 10 ng/mL (R&D Systems, Minneapolis, MN), IL-6 at 10 ng/mL (R&D Systems), granulocyte-macrophage colony-stimulating factor (GM-CSF) at 10 ng/mL (R&D Systems), stem cell factor (SCF) at 100 ng/mL (R&D Systems), and FLT-3 ligand (FLT-3L) at 100 ng/mL (R&D Systems). Cultures were replenished twice weekly by replacing half of the medium and cytokines and expanded into additional culture wells with or without pre-established PMVEC monolayers as required. At days 7, 14, and 21, aliquots of cells were harvested for the performance of cell counts, phenotypic analysis, and in vivo assays. The trypan blue exclusion method was used to determine the total viable cell content of expansion cultures. The large size and distinct appearance of PMVECs permitted their exclusion during cell counting and flow cytometric analysis.Phenotypic characterization and human lymphocyte antigen-typing of expanded and nonexpanded cells Primary CB and ABM cells and cells expanded ex vivo were phenotypically analyzed. Cells were preincubated with 1 mg/mL of human gamma globulin in staining buffer to block nonspecific binding. To determine the CD34+ and CD34+CD38
cell content of each sample, cells were then incubated with
phycoerythrin (PE)-conjugated monoclonal antibody against CD38 (Becton
Dickinson, San Jose, CA) and fluorescein isothiocyanate
(FITC)-conjugated monoclonal antibodies against CD34 (Becton
Dickinson). A portion of each sample was incubated with the appropriate
PE- and FITC-conjugated isotype control antibodies to establish the
background level of nonspecific staining. To determine HLA allele
expression, samples were incubated with BB7.2, GAP A3, MA2.1, BB7.1,
and MB40.2 monoclonal antibodies derived from ATCC hybridomas (ATCC,
Rockville, MD) or appropriate isotype control antibodies followed by
the incubation with PE-conjugated rabbit antimouse (H+L) (Zymed, South
San Francisco, CA). All staining was performed in dPBS staining buffer
supplemented with 2% FBS, 10 U/mL preservative-free heparin, and 1 mg/mL human gamma globulin. Propidium iodide (PI; 1 µg/mL) (Sigma)
was used during analysis to identify and exclude dead cells. Flow
cytometric analysis was performed using a FACSCalibur cytometer (Becton
Dickinson). At least 10 000 PI-negative events were collected.
Acquired data were analyzed using CELLQuest 3.1 software
(Becton Dickinson).
Triple mismatched SCID-human bone model The marrow repopulating potential of primary CB and ABM as well as corresponding ex vivo expanded cells was assessed using an in vivo triple HLA-mismatched SCID-human (SCID-hu) bone assay system44,45 (Figure 1). SCID-hu bone mice were constructed as previously described.42,43 Briefly, human fetal bone fragments obtained from elective abortions (Advanced Bioscience Resources, Alameda, CA) were subcutaneously implanted in 6- to 8-week old C.B-17/Icr-scid mice (Charles River Laboratories, Wilmington, MA) and allowed to vascularize for 8 weeks (SCID-hu bone mice). A small portion of human fetal bone marrow was reserved for HLA typing. Primary ABM and CB cells, expanded ABM, or expanded CB were individually or in various combinations injected into fetal bone grafts after the mice received 450 cGy of total body irradiation. Tissues injected into the same bone simultaneously differed in HLA allele expression from each other and the host bone. For all in vivo assays, different tissues were normalized for CD34+ cell content: equivalent numbers of CD34+ from each donor source were injected into each bone. To minimize the impact of tissue variability on the experimental data, various CB populations were tested as follows: half of the bone grafts, for example, were injected with the mixture of primary CB#1 and expanded CB#2 and the other half of bone grafts were injected with the mixture of primary CB#2 and similarly expanded CB#1. Each of the sources of HSCs was assayed in SCID-hu assays individually as well. Eight to 10 weeks after injection of the grafts, ABM cells were harvested from the fetal bone grafts and analyzed by flow cytometry for the hematopoietic contribution of the host and each injected donor. The presence of myeloid cells, lymphoid cells, and hematopoietic progenitor cells was detected with allophycocyanin (APC)-conjugated anti-CD33, CD19, and CD34 antibodies (Becton Dickinson), respectively. Donor and host cells were detected by a 5-color flow-cytometric assay using FITC, PE, and biotin-conjugated W6/32 (Leinco Technologies, St Louis, MO) and appropriate HLA allele monoclonal antibodies with FITC, PE, and biotin-conjugated goat antimouse IgG1, IgG2a, IgG2b (Southern Biotechnology Associates, Birmingham, AL) and streptavidin-conjugate PharRed (Pharmingen, San Diego, CA). Grafts were analyzed using FACSVantage flow cytometer. Immediately prior to analysis, 1 µg/mL PI was added for the identification and exclusion of dead cells. Uninjected grafts were used as controls for nonspecific staining. Grafts were considered positive if they contained more than 1% of cells expressing a particular donor HLA antigen.
Competitive repopulation of cord blood and marrow CD34+ cells Initially, we directly assessed the ability of 2 different sources of HSCs obtained at different times of ontogeny to competitively engraft and differentiate into multiple hematopoietic lineages within the same SCID-hu bone. Equal numbers (9 × 104) of ABM and CB CD34+ cells containing similar numbers of CD34+ CD38 cells bearing distinct HLA alleles
were directly injected individually and simultaneously into SCID-hu
bones. After 10 weeks the grafts were harvested and flow cytometrically
analyzed for the presence of ABM and CB progeny. As can be seen in
Table 1, when injected individually, both
ABM and CB CD34+ cells were each capable of producing
significant and comparable levels of multilineage engraftment in
virtually all fetal bones injected. However, when equal numbers of CB
and ABM CD34+ cells containing equal numbers of
CD34+CD38 cells were injected simultaneously
into the same fetal bone grafts, the CB and ABM behaved quite
differently. CB cells of multiple hematopoietic lineages were found in
every fetal bone injected; however, evidence of ABM engraftment was
present in only 50% of injected fetal bones. The percentage of
ABM-derived cells in those bones where engraftment was confirmed was
far less than the percentage of CB-derived cells in the same bones. In
addition, the degree of engraftment when only ABM CD34+
cells served as the graft was far greater than when the same dose of
ABM cells were cotransplanted with CB cells. CB CD34+ cells
were able to outcompete ABM CD34+ cells in this competitive
repopulation assay. These data provide a direct demonstration of the
ontogeny-related changes in human HSCs that had been previously
inferred from the behavior of purified stem cell population in a
variety of in vitro and in vivo assay systems.4-7,32,33
Competitive repopulation of primary and expanded cord blood grafts Recently, a number of laboratories have demonstrated that ex vivo expanded HCS possess inferior engraftment capabilities as compared to primary stem cells.8-12 This defect has been attributed to either replicative senescence or acquisition of a cell cycle-related homing defect.8-12,14,15 We next attempted to directly demonstrate the functional properties of expanded and primary HSCs using the triple mismatched SCID-hu model. Cord blood CD34+ cells were expanded in a stroma-free system or a PMVEC-based system to which SCF, IL-3, GM-CSF, IL-6, and FLT-3L were added. The degree of expansion of CD34+ cells and the CD34+CD38 cells in each of these culture
systems after 7 to 21 days of incubation is shown in Figure
2. After 21 days of expansion in a PMVEC
expansion system, a 5-fold greater expansion of CD34+ cells
and over a 241-fold greater expansion of
CD34+CD38 cells was observed as compared to
the stroma-free cultures supplemented with a similar cytokine
combination.
We then compared the engraftment capacity of primary CB cells and CB
CD34+ cells expanded in a PMVEC coculture system or in a
stroma-free culture system. The grafts were normalized prior to
injection into the triple HLA mismatched SCID-hu bone assay so as to
contain equal numbers of CD34+ cells
(1.5 × 104 cells/tissue), and donor contribution was
determined by flow cytomety 10 weeks later. Both primary CB cells and
CB cells expanded in either culture system engrafted in a similar
fashion; when injected individually each was able to establish a
significant degree of donor-derived multilineage hematopoiesis in
virtually all injected fetal bones (Tables
2 and 3).
However, when the primary CB cells and the PMVEC expanded CB cells were
injected into the same fetal bone grafts, the primary CB cells clearly outcompeted the PMVEC expanded CB cells (Table 2). It is important to
note that the primary CB grafts contained 50% of the number of the
CD34+CD38
We next tested the competitive repopulating potential of CB
CD34+ cells that had been expanded in either the
PMVEC-based or stroma-free expansion systems (Table
4). Both expansion products, when
transplanted alone, successfully engrafted all injected human fetal
bones and were capable of generating significant levels of
donor-derived CD34+, CD19+, and
CD33+ cells. However, when these 2 expansion products
containing equal numbers of CD34+ cells
(1.5 × 104 cells) were cotransplanted into the same
fetal bones, to our surprise, the cells produced in the stroma-free
system were present in 40% more fetal bones than the cells expanded in
the PMVEC cocultures (Table 4). Both expanded products, however,
clearly, retained the ability to differentiate into multiple
hematopoietic lineages (Figure 3).
These findings are somewhat surprising because the PMVEC expanded
grafts contained significant numbers of
CD34+CD38
Competitive repopulation of expanded cord blood and adult bone marrow grafts The competitive repopulating ability of CB cells expanded for 17 days in PMVEC cocultures supplemented with IL3, IL6, GM-CSF, SCF, and FLT-3L was compared to that of ABM expanded under the identical conditions. Immunomagnetically reisolated CD34+ cells (1 × 104) from HLA-mismatched CB and ABM were simultaneously injected into the SCID-hu bone assay. The CB expanded grafts contained 5-fold greater numbers of CD34+CD38 cells than the ABM expanded grafts
(Table 5). When injected individually, expanded ABM and CB cells each
demonstrated a significant level of multilineage donor engraftment in
all fetal bone grafts (Table 5). When
identical numbers of ABM and CB PMVEC coculture expanded cells were
assayed within the same SCID-hu bone graft, expanded CB cells
outcompeted expanded ABM cells; none of the injected grafts contained
ABM-derived cells, whereas each graft contained CB-derived multilineage
cells, albeit at a somewhat lower level than observed when primary CB
cells were assayed.
The direct evaluation of the repopulating potential of different ontogeny-related sources of murine HSCs has been facilitated by the use of competitive repopulation assays, which measure stem cell activity based on the demonstrated ability of cell populations to reconstitute hematopoiesis when transplanted into lethally irradiated recipients in competition with a defined population of cotransplanted cells that allow for the survival of the recipients receiving myeloablative radiation.34-39 The HSCs detected are termed competitive repopulating units (CRU) and have been quantitated in unseparated or purified donor cell populations by limiting dilution analysis techniques.34 The stem cell populations of interest can be tracked either by the expression of phenotypic markers or by retroviral gene marking. Because the genetic modification of HSCs by itself might potentially affect stem cell function, the use of phenotypic markers such as cell surface antigens, expression of intracellular isoenzymes, or hemoglobulin subtypes to track HSC progeny has permitted the identification of the progeny of different stem cell sources with this assay system.34-40 Such studies have demonstrated important ontogenic differences in HSC behavior.36-38 Murine fetal liver HSCs possess a greater proliferative activity in vivo than ABM stem cells.36,38 Furthermore, the functional activity of HSCs from late fetal and newborn mice, which is similar to CB in humans, has also been compared to murine peripheral blood and marrow stem cells using competitive repopulation assays.37 The repopulating potential of late fetal or newborn blood has been reported to be several times less than that found in a similar number of ABM cells, but far more than in normal adult blood.37 Direct assessment of the competitive repopulating potential of various sources of human HSCs has not been previously possible. A variety of in vitro and in vivo assays have, however, been used to assess the fundamental properties of human adult ABM and CB HSCs.29,31-33 CB progenitor cells have a higher in vitro plating and replating efficacy, which has been suggested to be a consequence of the greater self-renewal capacity of CB HSCs as compared to ABM HSCs.7,26,27,32 In addition, several xenogeneic animal systems using various murine models such as SCID, nonobese diabetic (NOD)/SCID, bg/nu/xid (BND) mice as well as an in utero sheep transplant model have been used for these same purposes.24,28,29,32,46-49 Transplanted human HSCs engraft in each of these models within a xenogeneic microenvironment. Use of these in vivo models has provided important information about the biologic properties of CB- and ABM-derived HSCs. CB, unlike adult HSCs, for instance, are capable of engrafting NOD/SCID mice without the administration of exogenous cytokines.28 To establish a competitive repopulation assay for human HSCs, we used a SCID-hu bone model in which human fetal bone fragments are implanted in the SCID mouse.44,45 The SCID-hu bone model permits assessment of the engraftment potential of a putative stem cell population within an appropriate human ABM microenvironment without the need for exogenous cytokines or carrier cells. The triple HLA-mismatched SCID-hu bone model is largely patterned after the competitive repopulation assay in mice.34-37 HSCs from 2 HLA-mismatched donors are injected into the same recipient and myeloid (CD33+), lymphoid (CD19+), and progenitor (CD34+) cells derived from each donor are identified after 8 to 10 weeks using antibodies specific for human major histocompatibility complex (MHC) class I alleles. The hematopoietic cells produced by each of the 2 grafts are not only competing with each other but also with cells produced from the endogenous fetal marrow that had previously been suppressed with sublethal irradiation. Equal numbers of CD34+ cells from the various ontogenic sources of HSCs served as grafts in cells to evaluate their relative proliferative capacity. The relative efficiency of engraftment and multilineage differentiation was judged by the number of grafts containing cells from each individual graft, and their ability to differentiate into multiple hematopoietic lineages. The triple HLA-mismatched SCID-hu model as presently described represents a qualitative assay of HSC function rather than a quantitative assay of HSC CRU. This assay, however, if performed in limiting dilution assay would be capable of generating a quantitative assessment of CRU as has been previously reported with the NOD/SCID stem cell assay.30 Engraftment is believed to involve the directed movement of HSCs to specific microenvironmental "niches" that favor HSC self-renewal and differentiation.50 At least in the marrow microenvironment of a fetal bone, CB stem cells appear clearly to have an advantage over ABM HSCs in occupying such theoretical "niches" and sustaining hematopoiesis for over 10 weeks. Because CB HSCs appear to have superior engraftment capabilities in comparison to ABM HSCs, the delayed engraftment patterns that are observed during clinical CB allogeneic transplantation appear not to be due to a qualitative defect of individual CB stem cells, but rather due to the infusion of a graft containing inadequate numbers of HSCs that are actually functionally more effective than ABM HSCs. This hypothesis is supported by Harrison and Astle who reported that the concentration of murine marrow repopulating cells were 2 to 4 times higher in ABM than in late fetal or newborn blood.37 Our studies imply that an effective strategy for overcoming the engraftment problems encountered with clinical CB transplantation is the infusion of increased numbers of these functionally superior CB HSCs. Strategies involving infusion of multiple CB grafts into a single recipient or the ex vivo expansion of CB stem cells are currently being explored to overcome these difficulties.51,52 Although the expansion of adult marrow repopulating stem cells has been reported to be clearly favored by cocultivation with endothelial cells, the optimal culture system with which to expand CB CD34+ has not been yet defined. To our surprise, CB CD34+ cells expanded in the stroma-free system outcompeted equivalent numbers of CB CD34+ cells with an identical cytokine combination expanded in the PMVEC coculture system when coinfused in the triple HLA-mismatched SCID-hu assay system. These findings emphasize the unique properties of the different ontogenic sources of HSCs that apparently have an impact on their potential for ex vivo expansion. The observation that expanded CB had a competitive repopulating advantage as compared to expanded ABM does, however, indicate that CB represents a more viable target for ex vivo stem cell expansion than ABM. In this report we describe an in vivo assay system that will hopefully be useful not only in examining the behavior of different sources of stem cells but also in defining improved strategies for genetic modification or expansion of HSCs with retention of their marrow repopulating potential. Correlation between the behavior of HSC sources in the triple HLA-mismatched SCID-hu bone assay and the behavior of comparable cell populations following transplantation in myeloablated large animal models will be required to evaluate this assay system as a surrogate human HSC assay.
The authors would like to thank the staff of Advanced Bioscience Resources Inc for their assistance in obtaining tissues for the construction of the model, and Judy Schnell and Priscilla Fitting for their assistance with flow cytometry.
Submitted May 1, 2000; accepted July 19, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ronald Hoffman, Eileen Heidrick Professor of Oncology, Chief, Section of Hematology-Oncology, University of Illinois at Chicago, 900 South Ashland Avenue, M/C 734, Chicago, IL 60607; e-mail: ronhoff{at}uic.edu.
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Abstract
Introduction
)
or stroma-free cultures (
) supplemented with recombinant human IL3,
IL6, GM-CSF, SCF, and FLT-3L. At days 7, 14, and 21, CD34+ (A) and CD34+CD38
at day 21 PMVEC cocultures contain 5 times more
CD34+ cells than stroma-free cultures. (B) No expansion
of CD34+CD38
and CD34+ CD38+ human hematopoietic progenitors from fetal liver, cord blood, and adult bone marrow respond differently to hematopoietic cytokines depending on the ontogenic source.
Exp Hematol.
1998;26:1034-1042