The GOD of hematopoietic stem cells: a clonal diversity model of the stem cell compartment.

Hematopoietic stem cells (HSC) show heterogeneous behavior even when isolated as phenotypically homogeneous populations. The cellular and molecular mechanisms that control the generation of diversity (GOD) in the HSC compartment are not well understood, but have been the focus of much debate. There is increasing evidence that the most important HSC functions, self-renewal and differentiation, are epigenetically preprogrammed and therefore predictable. Indeed, recent data show that the adult HSC compartment consists of a limited number of functionally distinct subsets of HSC. This contradicts older models of HSC behavior, which postulated a single type of HSC that can be continuously molded into different subtypes of HSC. We propose a clonal diversity model where the adult HSC compartment consists of a fixed number of different types of HSC, each with epigenetically preprogrammed behavior. Aging or disease may change the overall function of the HSC population. The model predicts that these changes reflect the relative composition of the HSC subsets, rather than changes in individual HSC. This view has implications for using HSC in experimental and clinical settings. Selection for the appropriate subsets of HSC, rather than attempts to force HSC to adjust, should improve their utility in transplantation and gene transfer applications.

Primitive hematopoietic stem cell function in vivo is uniquely high in the CXB-12 mouse strain.

Bone marrow cells (BMCs) from CXB-12/HiaJ (CXB-12) mice had 14 times the total long-term repopulating ability found in the best of 11 other CXB recombinant inbred (RI) lines. BMCs from each RI line donor were mixed with genetically marked standard competitor BMCs from the BALB/cByxC57BL/6 F1 (CByB6F1) hybrid, the mice used to produce the RI lines, and the mixtures repopulated lethally irradiated CByB6F1 recipients. Percentages of donor-type erythrocytes and lymphocytes measured the actual long-term repopulating functions of the donor RI lines relative to the standard competitor. CXB-12 BMCs repopulated better after 3 or 6 months than after 1 month, suggesting that the most primitive precursors were involved. Compared to CByB6F1 standard competitor cells, CXB-12 cells repopulated 3 to 12 times as well, with their advantage increasing when higher doses of cells were transplanted, probably because of hybrid resistance of the recipient against low doses. This was far better than expected, because F1 cells normally function 2 to 3 times as well as cells from an inbred strain. In competitive dilution, the advantage resulted from 2 factors: more precursor cells and more function per precursor. In the model that best fit the data, CXB-12 donors had 2.4 times the concentration of hematopoietic stem cells (HSCs) as the CByB6F1 standard, and each HSC repopulated 1.4 times as well. CXB-12 mice did not have elevated erythrocyte and lymphocyte numbers in blood and marrow and did not have unusually elevated concentrations of colony-forming unit spleen, cobblestone colonies, and long-term colony-initiating cells in marrow.

Tissue- and epitope-specific mechanisms account for the diverse effects of anti-CD44 antibodies on the maintenance of primitive hematopoietic progenitors in vitro.

The identification of rare stromal cells that support high levels of stem cells has opened avenues to identify molecules that contribute to the maintenance of these cells. We show that the maintenance of long-term culture initiating cells (LTC-IC) in stromal cell-supported cultures can be modulated via mAbs specific for CD44. mAb IM7.8.1 suppressed while mAb RAMBM44 enhanced LTC-IC levels in culture. Genetic polymorphisms in CD44 were used to show that the stromal cell compartment is targeted by mAb RAMBM44 and the hematopoietic compartment by mAb IM7.8. Neither of the CD44-specific mAbs inhibited adhesion of LTC-IC to the stroma, suggesting alternative mechanisms of action. In support of this interpretation, we show that mAb RAMBM44 directly induces signal transduction in the stromal cell line S17 but not in hematopoietic cells. Conversely, mAb IM7.8 elicited the appearance of phosphorylated bands in hematopoietic cells, but not in stromal cells. Collectively, the data indicate that the opposing effects of CD44-mediated regulation can be explained by different cellular programs that are elicited in distinct cell compartments. The binding of the enhancing mAb RAMBM44 to CD44 is specifically inhibited by collagen IV, while binding of the suppressive mAb IM7.8.1 is inhibited by a substance contained in the supernatant of the stromal cell line AC3.U. Thus, the CD44 epitopes defined by the mAbs bind distinct ligands and the ligands provide a potential physiological counterpart for the regulatory actions of the mAbs.

High frequency of long-term culture-initiating cells retain in vivo repopulation and self-renewal capacity.

OBJECTIVE: We wished to test if the long-term culture initiating cell (LTC-IC) assay measures primitive hematopoietic stem cells. An LTC-IC is defined by its ability to repopulate a stromal layer by forming colonies of myeloid cells. A negative well should never have received a stem cell, whereas a positive well should have been initiated by a stem cell. If these colonies were derived from stem cells, then a subset of the positive wells should retain stem cell activity. MATERIALS AND METHODS: Limiting dilution cultures were initiated on the stromal cell line S17. Individual clonal cultures from LTC-IC assays were assessed for repopulation capacity in W(14)W(41) mice. RESULTS: In long-term repopulation experiments, little activity was found in the negative wells, whereas 50% of the positive wells contained repopulating stem cells. The diverse in vivo repopulation patterns of the clonally derived stem cells suggest that this assay detects the full spectrum of stem cell types. Secondary transfers show that the clonally derived stem cells have self-renewal capacity. Experiments with mixtures of genetically distinguished cells showed that most (>90%) of the cultures were clonal. CONCLUSIONS: Our data present the first formal link between LTC-IC and repopulating stem cells. Moreover, the culture system presents a new way of generating a high frequency of clonally repopulating stem cells.

Genetic control of hematopoietic stem cell frequency in mice is mostly cell autonomous.

Previously we reported that the size of the stem cell compartment (measured as LTC-IC) is 11-fold greater in DBA/2 than in C57BL/6 mice, and we identified genes that regulate the size of the stem cell pool. To determine whether stem cell intrinsic or extrinsic events account for these differences, we created chimeras by aggregating morulae from the strains C57BL/6 and DBA/2. In these chimeras stem cells of both genotypes are exposed to a common mixed environment. Thus, an equalization of stem cell frequencies is expected if stem cell extrinsic effects dominate. Conversely, the parental ratio of LTC-IC should be preserved if the regulation is stem cell autonomous. For each chimera, individual LTC-IC were genotyped on the clonal levels by analyzing their progeny. We found that most of the difference that regulates the size of the stem cell compartment was intrinsic.

Bone marrow transplantation does not ameliorate the neurologic symptoms in mice deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT).

The use of bone marrow transplantation (BMT) for the treatment of genetic diseases with neurologic involvement has yielded mixed results. We have employed a mouse model of Lesch-Nyhan disease (LND) to assess the efficacy of BMT in ameliorating the neurologic manifestations of the disease. Adult HPRT-deficient mice exhibit a measurable decrease in striatal dopamine levels and a hypersensitivity to amphetamine. Marrow-ablated adult HPRT-deficient mice were transplanted with marrow from congenic HPRT-expressing mice. BMT altered neither the neurochemical nor the behavioral phenotypes in either HPRT-positive or HPRT-deficient mice. Barring any important species differences, these results suggest that BMT in its present form may not be an effective therapy for Lesch-Nyhan syndrome.

Genetic control of the frequency of hematopoietic stem cells in mice: mapping of a candidate locus to chromosome 1.

The genetic elements that govern the differentiation and proliferation of hematopoietic stem cells remain to be defined. We describe here marked strain-specific differences in the frequency of long-term culture-initiating cells (LTC-IC) in the bone marrow of different strains of mice. Mice of C57Bl/6 background showed the lowest levels of stem cells in marrow, averaging 2.4 +/- .06 LTC-IC/10(5) cells, BALB/c is intermediate (9.1 +/- 4.2/10(5) cells), and DBA/2 mice contained a 11-fold higher frequency of LTC-IC (28.1 +/- 16.5/10(5) cells) than C57Bl/6 mice. The genetic factors affecting the size of the stem cell pool were analyzed in the C57Bl/6 X DBA/2 recombinant inbred strains; LTC-IC frequencies ranged widely, indicating that stem cell frequencies are controlled by multiple genes. Quantitative trait linkage analysis suggested that two loci that have major quantitative effects are located on chromosome 1 near Adprp and Acrg, respectively. The mapping of the locus near Adprp was confirmed by finding an elevated stem cell frequency in B6.C-H25, a C57Bl/6 congenic strain that carries a portion of chromosome 1 derived from BALB/c mice. We have named this gene Scfr1 (stem cell frequency regulator 1). The allelic forms of this gene may be an important predictor of stem cell number and thus would be useful for evaluating cell sources in clinical stem cell transplantation.

Identification of a growth factor for primary murine stroma as macrophage colony-stimulating factor.

Knowledge of the stromal microenvironment is crucial for understanding the hematopoietic system. We took advantage of an assay that permits analysis of primary stroma-initiating cells (SICs) on the clonal level, and further characterized SICs and the factors that regulate SICs. Stroma formation in this assay is dependent on a high-molecular-weight factor secreted by the stromal cell line AC3.U. Here we show that this factor is identical to macrophage colony-stimulating factor (M-CSF), and that purified M-CSF is sufficient for induction of stroma formation. M-CSF, isolated from the line AC3.U, as well as from L929 cells and COS cells transfected with an expression vector encoding M-CSF, migrated in two peaks as 160- and 650-kD species after gel filtration. These molecular-weight species encompassed all stroma-inducing activity, and both stimulated macrophage colony formation. Affinity chromatography and blocking studies with antibodies specific for M-CSF and c-fms confirmed M-CSF as the sole factor in the supernatant of the stromal cell line AC3.U that promotes stroma formation. Culture of marrow, for as little as 1 week, depleted M-CSF-dependent SIC while increasing the incidence of replatable, factor-independent SIC. This suggests that culture changes the properties of SICs, perhaps by inducing differentiation into mature stromal cells. Thus, our results show a novel function of M-CSF as an important modulator of stroma formation.

The stromal cells' guide to the stem cell universe.

Pluripotent hematopoietic stem cells have emerged as a heterogeneous population of cells that differ in phenotype and repopulation kinetics. Stem cells in vitro and in vivo are dependent upon stromal cells for their proliferation and differentiation. Thus, stromal cells can be viewed as tools to analyze the physiological conditions that regulate stem cells. Stromal cell lines that support stem cells are infrequent, which supports the interpretation that stromal cells create distinct niches that regulate stem cell development. A model of stem cell maintenance is presented that predicts that stromal cell-bound molecules protect stem cells from differentiation. The stroma compartment is highly adaptable and can change its function in response to external stimuli. Thus, it is tempting to speculate that the stroma acts as a translator of peripheral signals for stem cells.

Clonal analysis of primary marrow stroma: functional homogeneity in support of lymphoid and myeloid cell lines and identification of positive and negative regulators.

Stromal cells play an important role in regulating early hematopoiesis. How stromal cells exert their different functions and the factors that regulate stromal cells themselves remain to be elucidated definitively, however. We describe here a limiting dilution assay for primary stroma colonies from murine marrow. This system permits a critical analysis of stromal cell function and regulation on the clonal level. We report that stroma formation was dependent on an activity secreted by the long-term cultured stromal line AC-3.U. Differential ultrafiltration of AC-3.U supernatant (SN) suggests that this potentially novel activity is represented by molecules with apparent molecular weights (m.w.) of > 100 < 300 kD and > 300 kD. In contrast to the AC-3.U activity, hydrocortisone (HC) acts as a negative regulator of stroma colony formation. We used the stroma colony assay to analyze potential stromal cell heterogeneity. We found that most, if not all, primary stromal colonies supported expansion of both myeloid and lymphoid cell lines. In contrast, long-term cultured stromal cell lines differed not only among lines, but also on the level of sublines, in their ability to sustain myeloid and lymphoid cells. This intraclonal variation suggests that the heterogeneity of cell lines can be a reflection of ongoing culture adaptation. The functional homogeneity of primary stromal colonies, together with their susceptibility to regulators, indicates that the performance of primary stroma is subject to external control. The establishment of a clonal assay system has paved the way to analyze the molecules that regulate primary stroma and thereby hematopoietic cells.

Stromal cells in long-term cultures: keys to the elucidation of hematopoietic development?

Long-term bone marrow cultures mirror many aspects of in vivo mammalian hematopoiesis. Thus, these cultures have been used widely to analyze the complex interactions that regulate hematopoietic differentiation. Hematopoiesis in vivo and in vitro is dependent on stromal cells, a mixture of support cells. In the past years numerous clonal stromal cell lines derived from murine and human tissues have been isolated and characterized. The stromal cell lines have proved to be invaluable tools for the elucidation of the molecular and cellular signals that govern differentiation and self-renewal of hematopoietic cells. This review describes the salient features of arrow cultures with a focus on the isolation and characterization of stromal cell lines. We summarize how stromal cell lines have been crucial tools for the detection, isolation, and maintenance of rare pluripotent stem cells and B lineage cells. Intriguing questions about the nature of stromal cells, their requirements for growth and differentiation, and their histogenic origin remain areas for further investigation.

Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit.

We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).

CD4 is expressed on murine pluripotent hematopoietic stem cells.

We show here for the first time that pluripotent hematopoietic stem cells express the CD4 antigen. CD4+ cells isolated from mouse marrow repopulated all hematopoietic lineages in both the long-term repopulation assay and the competitive repopulation assay. This finding indicates that the CD4+ population contains primitive stem cells with extensive repopulation capacity. Interestingly, the CD4- population had significant life-sparing activity, even though this population was depleted of long-term repopulating stem cells when compared with CD4+ cells. The majority of the cells that respond to the stroma in Whitlock-Witte cultures with B-cell differentiation were recovered in the CD4- population. Thus, this bone marrow (BM)-derived B-cell precursor lacks CD4, which is in contrast to myeloid precursors and thymus-derived lymphoid precursors that reportedly express CD4. We show further that the CD4 molecule expressed on BM cells is similar in molecular weight and epitope makeup to the CD4 antigen found on thymocytes. Detection of CD4 on BM cells is dependent on using high concentrations of antibodies. Thus, it is not surprising that expression of CD4 on pluripotent stem cells has been missed previously. Taken together, our data suggest that the CD4 molecule may play an important role in lineage definition in early hematopoietic differentiation.

Separation of pluripotent stem cells and early B lymphocyte precursors with antibody Fall-3.

A major goal in the study of hematopoiesis is to obtain populations of primitive stem cells, free of restricted and mature cells. We previously showed that a small population of normal bone marrow, the Thy-1loLin- cells, was highly enriched for pluripotent stem cells that repopulate lethally irradiated mice. These cells also differentiated along the B lymphocyte lineage in response to the stromal elements in Whitlock-Witte cultures. These two hematopoietic activities were entirely contained in and were enriched to similar extents in the Thy-1loLin- population. Here we show for the first time that these two activities can be resolved functionally and phenotypically. The cells that respond to the stroma in lymphoid culture are more sensitive to the cytotoxic drug 5-Fluorouracil than are stem cells. Furthermore, we have derived a new monoclonal antibody, Fall-3, that detects primitive stem cells but does not label the B cell precursor. This indicates that the small Thy-1loLin- population is heterogeneous, containing precursors restricted to the B cell lineage as well as pluripotent stem cells. Antibody Fall-3 defines a novel stem cell antigen, expressed on all primitive stem cells and thus, will be useful in the further characterization and isolation of both stem cells and B cell precursors.

Long-Term B Lymphoid Cultures from Murine Bone Marrow : Establishment and Cloning by Using Stromal Cell Line AC 6.21.

Nearly all hematopoietic cells in mammals derive from precursors that undergo much or all of their development in the bone marrow. In vitro models for many lineages are available and represent modifications of the original bone marrow culture system designed by Dexter and Lajtha (1) and described elsewhere in this book. In this chapter, we describe a second bone marrow culture system, first reported in 1982 (2), that provides an in vitro environment selectively supporting long-term proliferation and differentiation of early B lymphocyte lineage cells. This method can be used to obtain heterogeneous populations of immature precursors of the B cell lineage greatly enriched from other hematopoietic cell types. Clonal populations can also be obtained by extension of this method to limiting dilution culture.

Identification of a novel bone marrow-derived B-cell progenitor population that coexpresses B220 and Thy-1 and is highly enriched for Abelson leukemia virus targets.

A novel stage in early B-lymphocyte differentiation has been identified in normal mouse bone marrow cells. Earlier work had demonstrated that bone marrow cells characterized by low levels of Thy-1 and lack of a panel of lineage markers (Thy-1lo Lin- cells) were highly enriched for pluripotent hematopoietic stem cells. In this paper, we present evidence that another bone marrow population, which expressed low levels of Thy-1 and coexpressed B220, a B-lineage-specific form of the leukocyte common antigen, contained early and potent precursors for B lymphocytes upon in vivo transfer to irradiated hosts. These Thy-1lo B220+ cells, comprising 1 to 2% of bone marrow cells, were enriched for large cells in the mitotic cycle; the population lacked significant pluripotent hematopoietic stem cell activity and myeloid-erythroid progenitors. Most strikingly, Thy-1lo B220+ cells represented a highly enriched population of bone marrow cells that could be targets of Abelson murine leukemia virus transformation. We propose that Thy-1lo B220+ bone marrow cells represent the earliest stage of committed lymphocyte progenitors, intermediate in differentiation between Thy-1lo Lin- pluripotent stem cells and, in the B lineage, Thy-1- B220+ pre-B cells.