Assay systems for hematopoietic stem and progenitor cells

Assay Systems for Hematopoietic Stem and Progenitor Cells

THOMAS A. BOCK

Department of Hematology and Oncology, University of Tiibingen, Tiibingen, Germany

Key Words. In vitro assays . I n vivo assays . Hemntopoietic stem cells Henzatopoietic progenitor cells Long-tern7 culture ' Immunodeficient mice Fetal sheep

ABSTRACT Progress in our understanding of the

hematopoietic system as well as novel cellular and molecular biology techniques are increasing- ly promoting the ex vivo manipulation and ther- apeutic use of hematopoietic stem and progenitor cells. For both, development of stem cell therapies and basic hematopoietic research, test systems for hematopoietic stem cells are required to mon- itor the intrinsic and ex vivo-induced properties of these cells.

In vitro assays for primitive hematopoietic cells (colony-forming units-blast, cobblestone area-forming cells, long-term culture-initiating cells [LTC-IC]) have been established which demonstrate the proliferative and differentiation capacities of these populations. The potentials of these assays have been recently enhanced by the extended LTC and the switch LTC modifca- tions. Although some hematopoietic cells characterized in vitro have the multipotential and

proliferative properties of pluripotent hematopoietic stem cells (PHSC), their capacity to long-term repopulate hematopoiesis in vivo, a hallmark of PHSC, has not been established. Without this confirmation, populations defined in vitro should not be considered the equivalent of PHSC.

In animals, the properties of primitive hematopoietic cells can be systematically analyzed by multiple in vivo assays. Therefore, various strategies have been pursued to develop an animal model for human hematopoiesis. In fetal sheep and immunodeficient mice, the functions of human PHSC are reproduced, and long-term multilineage repopulation capacity and extensive proliferative potential have been demonstrated for xenografted human cells. Thus, both models can be considered stem cell assays and may significantly enhance the study of early hematopoiesis and the development of therapeu- tic strategies. Stem Cells 1997:15(supp1 1):185-195

Recent studies of early hematopoiesis have expanded our knowledge of the phenotypic properties, biology and regulation of hematopoietic stem cells. Phenotypic characterization of the most primitive pluripotent hematopoietic stem cells (PHSC), however, is still not discriminative enough to purify these cells to homogeneity. One reason for this is that surface markers on early hematopoietic stem cells have not yet been completely identified. Also, antigen expression, as well as other properties, are not specific for a singular hematopoietic cell type. There is considerable overlap in phenotype for cell morphology and surface markers between different cell types. Therefore, only a combination of multiple markers and characteristics could unequivocally define a PHSC, if at all. Furthermore, different stem cell phenotypes probably exist that are related to different functional states of these cells. As a consequence, even a pure population of stem cells may contain different phenotypes.

Hematopoietic Stem Cells. STEM CELLS 1997;15(suppl 1):185-195 0 1997 AlphaMed Press. All rights reserved.

186 Stem Cell Assays

Because of these current limitations in identifying a PHSC by its phenotype alone, functional in vivo and in vitro assays have been developed to indirectly demonstrate the presence of primitive hematopoietic cells in a heterogenous test population by determining the biologic functions of these cells. PHSC have been defined as cells with the biological properties of self-renewal and long-term repopulation of all lym- phohematopoietic lineages in a transplant recipient. Therefore, each assay attempts to define conditions under which early hematopoietic cells can exhibit those biological capacities. If those have been suffi- ciently demonstrated, then the elusive cell exhibiting these capacities can be considered the equivalent of a PHSC. Because of the indirect nature of these approaches, demonstration of presence of a specific cell type does not imply, however, that this cell can be identified as an individual cell of the input population, unless the test population contains only one single cell.

Multilineage differentiation is only indicative of PHSC when originating from one single ancestral cell as opposed to several single lineage-committed progenitors. Confirmation of clonal hematopoiesis either requires single cell studies or analyses of genetic markers within different lineages [l , 21. These analyses are elaborate and have not always been included in studies of stem cells. Instead, it has been assumed that committed progenitors are only short-lived because of their limited proliferative potential and because they die in differentiation. Thus, the presence of stem cells has been deduced from the demonstration of ongoing growth after several weeks.

It has been widely debated whether self-renewal, defined as the potential of giving rise to identical daughter cells, actually exists. Restrictions in serial transfer potential and telomer shortening during differentiation and ontogeny of hematopoietic cells both suggest that differences in proliferative potential within stem cell compartments exist [3,4]. However, if all daughter cells must differ in some respect from their parents, then the stem cell compartment would be extremely heterogeneous and hierarchical. The experimental demonstration of extensive proliferative potential includes the in vitro replating potential or the in vivo serial transfer capacity of hematopoietic cells, but this experimental evidence is not sufficient to settle this issue.

According to the above definition of a PHSC, lifelong monitoring of multilineage hematopoietic repopulation in a transplanted animal or person constitutes the golden standard for PHSC assays. Consequently, results from alternative assays obviously must comply with in vivo repopulation studies. However, demonstration of complete and stable hematopoietic reconstitution requires a period of months or years. To facilitate the study of early hematopoietic cells, many strategies have been developed for assaying these cells in vivo and in vitro over shorter time periods. However, none of these assays fully satisfies the criteria for PHSC analysis [5 , 61. Rather, it appears that the cells studied in these assays do not achieve the same degree of self-renewal and range of differentiation as they do in vivo.

Other obstacles include the technical and quantitative limitations of stem and progenitor cell assays. The poorer our knowledge of PHSCs, the larger and more heterogeneous the input population must be chosen and the more unwanted nonstem cells are contained. High numbers of contaminating cells, however, may obscure the effects of the stem cells. Also, PHSC could potentially contaminate the selected test cells, read out in the assay and cause a false positive result.

On the other hand, the greater our knowledge a priori, the earlier hematopoietic cells can be enriched. As a consequence, very few cells are contained in the test samples and techniques may not be sensitive enough to assay such a small number of cells. There is a high risk of losing these cells during processing and transfer resulting in false negative results. This particularly concerns in vivo assays, which have relatively low seeding efficiencies for transplanted cells (-20%). In addition, highly enriched test cells are deprived of accessory cells which may be necessary for their proper functions. Therefore, accessory cells or their signals must be complemented.

In summary, in order to assess the validity of a PHSC assay, it must be tested whether the assay para- meters provide sufficient evidence for clonal multilineage differentiation, extensive proliferative potential and repopulation ability. In addition, technical issues have to be addressed due to the multiplicity of existing procedures and methods which are presented below.

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DIRECT CLONOGENIC ASSAYS Clonogenic assays are the most direct quantitative means of measuring hematopoietic cells by colony

formation in vitro. Hematopoietic colonies are clones of cells produced by a single progenitor cell. Hence, the number of colony-forming progenitors is directly related to the number of colonies. By replating the cells into different clonogenic systems, information regarding self-renewal and the differentiation capacities of the colony-forming cell (CFC) is generated.

Blast Colony-Forming Cell Assay (CFC-Blast) Ogawa has described a clonogenic assay for CFC-blast which detects a 5-fluorouracil (5-FU)-resistant

progenitor with secondary recloning potential and the capacity to generate single lineage and multilineage- committed progeny [7-121. The assay is defined for steady-state conditions and is based on the premise that the earliest hematopoietic cells are in Go. Bone marrow cells are plated in the presence of fetal calf serum, without complementary cytokines, under the assumptions that early cells are kept in Go and actively cycling progenitors are not supported. Colonies produced by CFC-blast consist of 50-500 immature hematopoietic cells. Their growth is delayed relative to the formation of granulocyte-macrophage colonies and erythroid bursts. However, the appeareance of colonies could be hastened by adding cytokines. Colonies produced by CFC-blast contained new CFC-blast which were detected by replating cells from the colonies under conditions that are identical to the primary culture. This reproduction capacity allows expansion of CFC-blast to relatively high numbers, but impairs reliable quantification of these cells. Alternative conditions for lineage commitment and differentiation can be used in the secondary culture in order to determine the functional spectrum of CFC-blast.

High Proliferative Potential-Colony-Forming Cells (HPP-CFC) HPP-CFC are characterized by their ability to generate very large colonies containing greater than

50,000 cells at 10-12 days of culture [13, 141. The frequency of HPP-CFC in normal marrow was reported at about 2 per lo5 mononuclear cells. HPP-CFC are resistant to 5-FU toxicity. They are made up of several subpopulations, suggesting a hierarchical organization within this progenitor cell compartment [ 14, 151. A portion of replated HPP-CFC-derived colonies contained further HPP-CFC, suggesting some self-renewal potential of HPP-CFC [ 161. The most primitive subsets have been described as being closely related with long-term reconstituting cells [13, 171. Murine, as well as human HPP-CFC, produced colonies which contained progenitors of granulocytes, erythrocytes and megakaryocytes [ 181.

Cobblestone Area-Forming Cell (CAFC) Assay When murine marrow is added to preformed stromal layers, colonies of adherent hematopoietic cells

(cobblestone areas) can he observed [ 191. Following up this observation in the human system, Gordon et al. [20] described a human cell type that adhered to stroma and produced colonies of undifferentiated blast cells. Replating of these colonies showed limited self-renewal and the presence of CFU-granulocyte-erythroid-macrophage-megakaryocyte (GEMM), CFU-GM and BFU-E, suggesting that CAFC are ancestral to CFU-GEMM [2 11. In the murine system, cells generating early-appearing cobblestone areas (days 7-10) appeared to coenrich with CFU-spleen and committed progenitors. On the other hand, cells forming late-appearing cobblestone areas (day 28) coenriched with cells providing short-term hematopoietic repopulation [22,23]. Although CAFC determined at weeks 5-8 correlated closely with in vivo repopulating ability [23], there is not sufficient evidence to support that CAFC represent PHSC [24].

In order to simplify the initial culture setup and eliminate inhomogeneities in the stromal feeder layer, fresh marrow-derived stromal layers were substituted with clonal cell lines. A murine bone marrow stromal cell line GB1/6 [25] and a preadipose cell line FBMD-l [26] were both found to support the growth of overlaid fresh marrow with the same efficiency as fresh stromal layers. For quantification of CAFC, Ploemacher described a long-term culture (LTC) system using microtiter wells. Cells are overlaid


at a limiting dilution on previously established irradiated stromal layers. The percentage of wells with at least one hematopoietic cobblestone area is then determined over a period of approximately 40 days, and CAFC frequencies can be calculated using Poisson statistics [23].

Frequency of CAFC in steady-state human marrow was determined at about 45 (week 5) and 15 (week 8) per lo5 Ficolled cells [26]. Murine CAFC frequency declined by over one log between days 7 and 28 with values of 2-40 per lo5 cells at day 28 [23, 251. Murine CAFC have been described as wheat germ agglutinin (WGA)d'm [27], and the phenotype of human CAFC was reported as CD34', HLA-DR-, Rh123dU" [26], 1% and Thy-1' [28].

SECONDARY CLONOGENIC ASSAYS This group includes assays for progenitor cells that do not themselves form colonies in semisolid

media but instead can produce clonogenic progeny. Test populations are cultured for several weeks and then analyzed for the number of clonogenic cells present.

LTC Assays Stromal cell-associated hematopoiesis has been extensively studied by Dexter and others as a means for

reproducing the in vivo environment for stem and progenitor cells [29-3 11. Several groups have established various LTC assays which detect an LTC-initiating cell (LTC-IC) [32-381. After five to eight weeks, primary stroma-supported cultures are scored for replatable colony-forming cells (CFC) that are released into the culture supernatant and assayed in semisolid culture systems. LTC can be set up either by inoculating tissue culture flasks with bone marrow, or alternatively, in two stages by growing the stromal layer to confluence and then adding hematopoietic cell suspensions. As in the CAFC assay, stromal cell lines have been employed to replace primary human marrow adherent feeder layers [39-411.

Since the LTC system is a qualitative method, frequency analysis of progenitor subsets is not feasible unless carried out as a limiting dilution approach. Eaves et al. [38] demonstrated a linear relationship between the number of cells used to seed a preformed stroma and the numbers of CFU-GM released at five weeks. However, individual LTC-IC produced variable numbers (1-30) of clones, and the number of LTC-IC continously declined with only a fraction persisting after five weeks [42]. Therefore, the absolute numbers of LTC-IC in different hematopoietic tissues cannot be compared unless the rate of decline is known for the test sample and corrected accordingly. Furthermore, long-term repopulating ability was lost in LTC of murine bone marrow 1431. Frequency of LTC-IC at weeks 5 and 8 was determined to be about 40 and 15, respectively, per 10' Percolled bone marrow cells, and represented about one percent of the CD34' cell population [36, 401. In peripheral blood, LTC-IC concentration was two logs lower and amounted to six cells per ml [44].

LTC-IC and CAFC share a series of functional and phenotypic properties. A considerable portion of both were resistant to cytotoxic drugs [45]. Both were preferentially, but not exclusively, contained in the CD34' Rhl 23dU" fraction of Ficolled bone marrow cells, and had a Thy-1' and HLA-DR'"" phenotype [40, 46-48]. LTC-IC were distributed between the CD38' and CD38- fractions and appeared to express low levels of c-kit [48, 491. Another study suggested that the c-kit' fraction contains the most primitive progenitor cells in human marrow [SO].

Crooks and colleagues extended the period of standard long-term stromal cocultures (supplemented with interleukin 3 [IL-31, IL-6 and stem cell factor [SCF]) to over 60 days [49, 511. They observed two peaks of CFC production, the first during days 35-60 and the second during days 60-100. Extended (E)LTC-IC were exclusively contained in the CD34'CD38- fraction and arose almost exclusively from late proliferating cells which formed visible clones after day 30. In contrast, standard LTC-IC arose from early proliferating cells between days 14-28 and were distributed between the CD38' and CD38- fractions. Clones that appeared after 60 days from single late proliferating cells contained tenfold more cells than from early proliferating cells. Whereas retroviral gene marking of LTC-IC showed efficient gene

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marking, ELTC-IC were not marked with vector. These studies suggest that ELTC-IC are a primitive progenitor population functionally distinct from LTC-IC.

Recently, a different modification of the LTC assay has been described by the Vancouver group [52,53]. It allows the identification and quantitation of a subset of murine LTC-IC (designated as LTC-ICML) with the capacity to generate both myeloid and B lymphoid progeny in vitro. Test cells are cultured at limiting dilu- tions on irradiated mouse marrow feeder layers for an initial four weeks under conditions supporting myelopoiesis and then for an additional week under conditions permissive for B lymphopoiesis. Clonogenic pre-B progenitors detected in such postswitch LTC appeared to be derived from uncommitted cells present in the original cell suspension. However, clonal myelolymphoid hematopoiesis was not analyzed. LTC-ICML were 5-F'U-resistant and coenriched 500-fold with LTC-IC in the SCA-l', lin-, WGA' fraction [52]. Their frequency was determined at approximately 1 per 5 x lo5 normal adult mouse bone marrow cells, which represented a 15-30-fold lower level than standard LTC-IC or cells with long-term in vivo myelolymphoid repopulation potential. Both standard LTC-IC and LTC-ICML were significantly better maintained in vitro than repopulating cells [53], suggesting differences in detectability or the developmental stages of these cells types.

Delta (A)-Assay A group of assay systems has become known as A-assays because they measure the production of

multiple clonogenic progeny (base of the A) by a single progenitor (apex of the A symbol). Delta assays permit analyses of recruitment, proliferation potential and lineage potentials of early hematopoietic cells. Bone marrow depleted of committed progenitors and enriched for primitive hematopoietic cells is grown in a seven-day suspension culture in the presence of various cytokine combinations. The effect of these cytokines on early progenitor cells is measured in a secondary clonogenic assay for committed progenitors [54, 551. Other variants of A assays utilize the properties of early hematopoietic cells to adhere to stroma [24] or plastic [56,57]. Cells producing CFU-GM in the latter assay modification have been described as 5-FU-resistant and as expressing CD34 and Thy-1 [56,58]. The cells detected by the different various A assays are heterogeneous but probably related and overlap with the LTC-IC [59].

IN VIVO XENOTRANSPLANTATION ASSAYS In vitro assays have greatly expanded our knowledge of the phenotype of primitive human progenitor cells

and their lineage relationships. Although some hematopoietic cells characterized in vitro have the multipotential and proliferative properties of PHSC, their capacity for long-term hematopoietic repopulation has not yet been established. Until this capacity has been confirmed, the populations defined by in vitro assays cannot be considered the equivalent of long-term repopulating PHSC. In animals, the properties of the most primitive hematopoietic cells can be systematically analyzed by multiple in vivo assays [ S , 60-631 which are not feasible in humans for ethical reasons. Consequently, various strategies have been pursued to develop an animal model for human hematopoiesis. Ideally, this model would provide the critical microenvironment for the engraftment of human hematopoietic cells without mediating rejection or permitting graft-versus-host-disease. Growth and differentiation of human cells would be supported and reflected by a physiological distribution of primitive and differentiated human cells in the host.

In the past few years, novel chimeric animal models have been developed that may significantly enhance our ability to analyze the functions of human PHSC in vivo, but outside of the human body. These models include xenotransplantation of human hematopoietic cells in immunodeficient mouse strains and preimmune fetal sheep. Recent results from these chimeric systems suggest that these animals provide a permissive environment for the study of human stem cells in vivo.

Fetal Sheep In sheep, long-term engraftment of human hematopoietic cells that have been injected into the peri-

toneal cavity of preimmune fetuses has been achieved [64-661. In utero transplanted cells from human


fetal liver, adult bone marrow and adult peripheral blood reconstituted myelolymphoid hematopoiesis, including T lymphocytes. Human cells could be monitored in all hematopoietic tissues and the peripheral blood for at least several months and up to several years [65-671. Engraftment was successful with as few as several hundred progenitor-enriched human bone marrow cells, and was restricted to the c-kirl"" as opposed to the c-kit' or c-kit- fractions [68]. Isolated CD45' human cells from the marrow of adult sheep, previously injected in utero with human fetal liver cells, could be transferred and engrafted to secondary sheep recipients [66]. However, the level of engraftment of human cells was still low in the primary sheep (3%-4%), and only two of six secondary recipients supported continued growth of human cells. Since myeloablative regimens are not feasible in pregnant sheep because of teratogenic hazards, xenografted donor cells are in competition with the endogenous pool of stem cells. Early engraftment of a certain percentage of donor cells occurs at a low proportion and stability cannot be fully achieved in the course of gestation. However, the engraftment level could be enhanced by ex vivo incubation of fetal liver cells with hematopoietic growth factors [67].

In order to establish a short-term assay for primitive hematopoietic cells, Zunjuni and coworkers transplanted human cells into the ovine fetus, sacrificed the fetal recipient two months later and transferred the recovered human CD45' cells into secondary recipients. Engraftment capacity in the secondary recipient (determined two months after secondary transfer) strictly correlated with the long-term multilineage hematopoietic reconstitution capacity (one to two years) of selected test populations in primary sheep (Zanjuni, personal communication). If confirmed, this modification may allow the identification of populations containing human long-term repopulating cells in a four-month assay.

Immunodeficient Mouse Models Xenotransplantation approaches would be facilitated by the development of simple transfer proce-

dures and models that are more economical than sheep. The identification of genetically immunodeficient mouse strains has provided a small animal model with a permissive microenvironment for the engraftment of human hematopoietic cells [69-7 I]. The most commonly used mouse strains include beige, nude and X-linked immunodeficient (bnx) mice, severe combined immunodeficient (SCID) mice and recently, nonobese diabetic (NOD-)KID mice.

Bnx mice were constructed by combining three recessive mutations which synergistically affect different cellular immune activities. The reduced immunity of the parental nude mouse (affecting T cell differentiation) was further reduced by the effects of the beige (cytotoxic T lymphocyte and natural killer [NK] cells) and X-linked immunodeficiency (lymphokine-activated killer cells and certain B cell responses) mutations [72-741. SCID mice are severely deficient in B and T lymphocytes, since lymphocyte differentiation is arrested as a result of a dysfunctional VDJ recombination of immunoglobulin and T cell receptor genes [70, 75, 761. However, residual immunity in both SCID and bnx mice, due to NK cells and macrophages, can mediate rejection of xenografted human cells [77]. To generate a SCID strain with low NK cell activity, the scid mutation was recently backcrossed onto the NOD/Lt strain background, which is characterized by a deficiency in NK cell activity, by functional defects of both antigen presenting cells and macrophages, and by reduced lytic com- plement activity [71]. NOD/Lt mice display a high incidence of autoimmune T cell-mediated diabetes melli- tus [78] which is prevented in NODLtSz-scidscid (NOD-SCID) mice due to the scid-related absence of mature T cells [7 1,791,

Engraftment of human hematopoietic cells has been achieved in all three mouse strains, and engraftment levels appeared highest in NOD-SCID mice [80-831. This may reflect the extensive immunodeficiency of this strain, but may also result from the particular support of human hematopoietic cells by NOD-SCID stre ma. Multiple strategies have been pursued to provide complementary support for proliferation and differentiation of xenografted cells. In the SCID-hu model, human fetal thymus or fetal bone fragments have been cotransplanted with human fetal liver cells in order to provide a human microenvironment [84-871. Alternatively, recombinant human (rHu)SCF and PIXY 321, a fusion protein of HuGM-CSF and HuIL-3,

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have been injected into SCID mice [88, 891. Selection of these cytokines was based on the rationale that SCF, GM-CSF and IL-3 act highly species-specific and promoted growth and differentiation of bone marrow progenitor cells in vitro [go]. In another approach, expression of the HuIL-3, GM-CSF and SCF transgenes enhanced the engraftment of umbilical cord blood or adult bone marrow cells in SCID mice [82]. Finally, human hematopoietic cells were cotransplanted into bnx mice with primary human bone marrow stromal cells engineered to produce HuIL-3 [SO].

Immunodeficient mice have been permissive to the engraftment of human hematopoietic cells from various sources including fetal liver, cord blood, adult bone marrow or adult peripheral blood. Xenografted cells differentiated into the myeloid, erythroid and B lymphoid lineages [83, 871. T lymphocytes were also reconstituted when human fetal thymus was cotransplanted with fetal donor cells [84, 911. Hematopoiesis was clonal as demonstrated by retroviral marking and persisted long-term for at least several months [2, 80, 82, 871. Donor cells recovered from human fetal bone grafts of SCID-hu mice eight weeks postinjection had the capacity to reconstitute myelolymphoid hematopoiesis in bone grafts of secondary SCID-hu recipients [87].

Using cell purification, retroviral gene marking and limiting dilution approaches, Dick and coworkers established that the cells capable of engrafting NOD-SCID mice are biologically distinct from LTC-IC and ancestral to these progenitors [83]. The frequency of the SCID-repopulating cell (SRC) was determined at about 1 per lo6 human cord blood and 1 per 3 x 10' bone marrow cells. SRC were exclusively CD34'CD38-. The attempts to retrovirally transduce these cells have been very inef- ficient [ 831.

SUMMARY The ultimate proof of PHSC requires demonstration of long-term multilineage hematopoietic repopula-

tion in a transplant recipient. Alternatively, short-term assays may be very useful and enhance the study of early hematopoiesis. This is particularly true for the human system. However, validity and specificity of alternative assays must be confirmed in vivo. This has either not been performed for most in vitro assay systems or results have not been satisfying [S, 921.

As a recent improvement, the LTC-ICML extends the range of differentiation that hematopoietic cells have achieved in other culture systems. Furthermore, the extended LTC system appears to detect a cell more primitive than standard in vitro-defined progenitors. The properties of the (E)LTC-IC cell, as far as they are known, are shared with the presumed PHSC phenotype.

The development of xenotransplantation strategies using immunodeficient mice or fetal sheep has result- ed in attractive models for human hematopoietic cells. Since long-term lymphomyeloid in vivo repopulation capacity, as well as extensive proliferative potential, have been demonstrated for the cells engrafting immunodeficient mice and preimmune fetal sheep, these xenograft models both appear to be promising assays for PHSC. However, additional progress could be helpful in establishing these systems as a better quantitative assay for xenorecipient repopulating-cells (XRC). Since quantitation often involves Poisson analysis of multiple limiting dilution setups, chimeric mice may be more useful than sheep. The coenrichment of cells exhibiting secondary transfer potential with long-term XRC supports the prospect that short-term in vivo assays for human hematopoietic stem cells are feasible.

However, until purification of PHSC to homogeneity is achieved and a specific PHSC phenotype is established, proof of PHSC requires the demonstration of long-term hematopoietic repopulation of all lineages in a transplant recipient. Only cells providing such evidence should be considered PHSC. Concern exists that differences in functional state or proliferative capacity of individual PHSC may result in extreme phenotypic heterogeneity and multiple stem cell subsets, and may challenge our ability to purify PHSC to homogeneity. However, only with a phenotypically homogeneous input population of PHSC can studies using different cell culture and animal models be considered truly comparative. Additionally, these studies may be affected by the limited assay sensitivity to minimal numbers of PHSC in a presumably homogeneous subpopulation of stem cells. In view of these many obstacles in studying


the most primitive hematopoietic cell, the isolation of the PHSC truly does earn the designation of the holy grad of hematology.

ACKNOWLEDGMENT

longstanding support as well as that of Dr. Lothar Kunz and Dr. Arthur W. Nienhuis. I am indebted to Dr. David M. Bodine and Dr. Donald Orlic, and gratefully acknowledge their

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Assay systems for hematopoietic stem and progenitor cells

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