Expression of interferon-gamma by stromal cells inhibits murine long-term repopulating hematopoietic stem cell activity.

Several lines of evidence suggest that overexpression of interferon gamma (IFN-gamma) in the marrow microenvironment may play a role in the pathogenesis of marrow suppression in aplastic anemia. We previously showed that overexpression of IFN-gamma by marrow stromal cells inhibits human long-term culture initiating cell activity assayed in vitro to a much greater degree than the addition of soluble IFN-gamma. The effect of IFN-gamma on true repopulating stem cells assayed in vivo has not been studied previously. We compared the effect of co-culture of murine marrow cells in the presence of stromal cells transduced with a retroviral vector expressing murine IFN-gamma vs stromal cells transduced with a control neo vector. Using a murine congenic competitive repopulation assay, there was significantly less long-term repopulating stem cell activity remaining after culture on mIFN-gamma-expressing stroma as compared to control stroma. We also investigated the effect of directly transducing murine bone marrow cells with the mIFN-gamma or control vector. Marrow cells transduced with either vector were transplanted into W/Wv recipient mice. The percentage of vector-containing cells in the mIFN-gamma mice was significantly lower than in the control mice, suggesting that mIFN-gamma-transduced primitive cells may not have survived culture, or that mIFN-gamma directly decreases gene transfer into repopulating cells. Despite no significant differences in white or red blood cells in the mice transplanted with the mIFN-gamma-transduced cells, the number of bone marrow colony-forming unit-C 16 weeks after transplantation was significantly lower in the IFN-gamma group. These data indicate that ectopic or overexpression of mIFN-gamma, especially by marrow microenvironmental elements, may have a marked effect on primitive hematopoiesis as assayed in vivo.     

All-trans retinoic acid enhances the long-term repopulating activity of cultured hematopoietic stem cells

The retinoic acid receptor (RAR) agonist, all-trans retinoic acid (ATRA), is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. We previously reported that pharmacologic levels (1 micromol) of ATRA enhanced the generation of colony-forming cell (CFC) and colony-forming unit-spleen (CFU-S) in liquid suspension cultures of lin- c-kit+ Sca-1+ murine hematopoietic precursors. In this study, we further investigated the effects of ATRA as well as an RAR antagonist, AGN 193109, on the generation of transplantable cells, including pre-CFU-S, short-term repopulating stem cells (STRCs), and long-term repopulating stem cells (LTRCs). ATRA enhanced the ex vivo maintenance and production of competitive repopulating STRCs and LTRCs from lin- c-kit+ Sca-1+ cells cultured in liquid suspension for 14 days. In addition, ATRA prevented the differentiation of these primitive stem cells into more mature pre-CFU-S during the 14 days of culture. In marked contrast, lin- c-kit+ Sca-1+ cells cultured with AGN 193109 for 7 days had virtually no short- or long-term repopulating ability, but displayed an approximately 6-fold increase in the pre-CFU-S population. The data suggest that the RAR agonist ATRA enhances the maintenance and self-renewal of short- and long-term repopulating stem cells. In contrast, the RAR antagonist AGN 193109 abrogates reconstituting ability, most likely by promoting the differentiation of the primitive stem cells. These results imply an important and unexpected role of retinoids in regulating hematopoietic stem cell differentiation. (Blood. 2000;95:470-477)     

Stem cell factor enhances proliferation, but not maturation, of murine megakaryocytic progenitors in serum-free culture.

The effects of recombinant rat stem cell factor (SCF/c-kit ligand) on murine megakaryocytopoiesis were studied using partially purified bone marrow cells derived from normal and 5-fluorouracil (5-FU)-treated mice in a serum-free culture system. SCF alone did not support the formation of megakaryocyte (M) and granulocyte-macrophage-megakaryocyte (GMM) colonies. However, the addition of SCF to cultures containing interleukin-3 (IL-3) resulted in a significant increase in the number of M and GMM colonies formed by bone marrow cells from normal mice, whereas IL-6 augmented only M colony growth. The stimulatory effect of SCF was approximately three to four times as high as that of IL-6 on the primitive progenitors capable of megakaryocytic-lineage expression derived from 5-FU-treated mice. In addition, SCF, but not IL-6, significantly increased the number of constituent cells in the individual M colonies supported by IL-3. On the other hand, SCF did not exert any effect on the size and DNA content of megakaryocytes in IL-3-dependent M and GMM colonies, whereas IL-6 enhanced the maturation of megakaryocytes. These results suggest that SCF stimulates the proliferative process in megakaryocytic progenitors and that the main activity of IL-6 is the promotion of megakaryocyte maturation.     

Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal

Although the wild-type prion protein (PrP) is abundant and widely expressed in various types of tissues and cells, its physiological function(s) remain unknown, and PrP knockout mice do not exhibit overt and undisputed phenotypes. Here we showed that PrP is expressed on the surface of several bone marrow cell populations successively enriched in long-term (LT) hematopoietic stem cells (HSCs) using flow cytometry analysis. Affinity purification of the PrP-positive and -negative fractions from these populations, followed by competitive bone marrow reconstitution assays, shows that all LT HSCs express PrP. HSCs from PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients both in the presence and absence of competitors. When treated with a cell cycle-specific myelotoxic agent, the animals reconstituted with PrP-null HSCs exhibit increased sensitivity to hematopoietic cell depletion. Ectopic expression of PrP in PrP-null bone marrow cells by retroviral infection rescued the defective hematopoietic engraftment during serial transplantation. Therefore, PrP is a marker for HSCs and supports their self-renewal.     

Long-term repopulating ability of telomerase-deficient murine hematopoietic stem cells

Telomere length must be tightly regulated in highly proliferative tissues, such as the lymphohematopoietic system. Under steady-state conditions, the levels and functionality of hematopoietic-committed or multipotent progenitors were not affected in late-generation telomerase-deficient mice (mTerc(-/-)) with critically short telomeres. Evaluation of self-renewal potential of mTerc(-/-) day-12 spleen colony-forming units demonstrated no alteration as compared with wildtype progenitors. However, the replating ability of mTerc(-/-) granulocyte-macrophage CFUs (CFU-GMs) was greatly reduced as compared with wildtype CFU-GMs, indicating a diminished capacity of late-generation mTerc(-/-) committed progenitors when forced to proliferate. Long-term bone marrow cultures of mTerc(-/-) bone marrow (BM) cells show a reduction in proliferative capacity; this defect can be mainly attributed to the hematopoietic, not to the stromal, mTerc(-/-) cells. In serial and competitive transplantations, mTerc(-/-) BM stem cells show reduced long-term repopulating capacity, concomitant with an increase in genetic instability compared with wildtype cells. Nevertheless, in competitive transplantations late-generation mTerc(-/-) precursors can occasionally overcome this proliferative impairment and reconstitute irradiated recipients. In summary, our results demonstrate that late-generation mTerc(-/-) BM cells with short telomeres, although exhibiting reduced proliferation ability and reduced long-term repopulating capacity, can still reconstitute myeloablated animals maintaining stem cell function.     

Stem cell factor induces proliferation and differentiation of highly enriched murine hematopoietic cells.

Recombinant rat stem cell factor (SCF) was studied for its ability to stimulate the growth of murine hematopoietic progenitor cells and to generate colony-forming cells (CFC) from highly enriched populations of hematopoietic cells. In serum-deprived cultures, SCF alone stimulated few colonies but interacted with a number of other hematopoietic growth factors, particularly interleukin 3, to promote colony formation. The most marked effect was on the generation of mixed-cell colonies. Hematopoietic cells were sorted into wheat-germ agglutinin-negative, monocyte-depleted, rhodamine 123 (Rh123)-bright or Rh123-dull cells. Historically, Rh123-bright cells are capable of short-term (less than 1 mo) marrow engraftment, whereas among Rh123-dull cells are cells capable of long-term marrow engraftment. Enriched cells (2.5 x 10(3) were placed into serum-deprived liquid cultures with various hematopoietic growth factors. Initially, the Rh123-bright and Rh123-dull cells had few CFC but, in the presence of interleukin 3 and SCF, Rh123-bright cells gave rise to greater than 15,000 granulocyte/macrophage CFC, greater than 1500 erythroid burst-forming cells, and greater than 700 mixed-cell CFC by day 5. In contrast, Rh123-dull cells proliferated only in the presence of interleukin 3 and SCF, but total cell numbers rose to a peak of 18,000 by day 21, and one-third of the cells were CFC. Thus, SCF, in combination with other growth factors, can generate large numbers of CFC from pre-CFC and appears to act earlier than hematopoietic growth factors described to date.     

Transforming growth factor beta 1 directly and reversibly inhibits the initial cell divisions of long-term repopulating hematopoietic stem cells

Hematopoiesis appears to be regulated, in part, by a balance between extracellular positive and negative growth signals. Transforming growth factor beta-1 (TGF-beta 1) has been shown to be a negative regulator of primitive hematopoietic cells. This study examined the direct effect of TGF-beta 1 on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC) in vitro. We previously reported a cell fractionation approach that includes the selection of low Hoescht 33342/low Rhodamine 123 (low Ho/Rh) cell fractions that are highly enriched for long-term repopulating cells (LTR-HSC) and also clone to a very high efficiency in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6: 90% to 100% of individually cultured low Ho/Rh cells formed high proliferative potential clones. This high cloning efficiency of an LTR-HSC enriched cell population enabled proliferation inhibition studies to be more easily interpreted. In this report, we show that the continuous presence of TGF-beta 1 directly inhibits the cell division of essentially all low Ho/Rh cells (in a dose-dependent manner) during their 0 to 5th cell division in vitro. Therefore, it follows that TGF-beta 1 must directly inhibit the proliferation of LTR-HSC contained within these low Ho/Rh cells. The time required for some low Ho/Rh cells to undergo their first cell division in vitro was also prolonged in the presence of TGF-beta 1. Furthermore, when low Ho/Rh cells were exposed to TFG-beta 1 for varying lengths of time before neutralization of the TGF-beta 1 by monoclonal antibody, the ability to form macroclones was markedly decreased after approximately 4 days of TGF-beta 1 exposure. In addition, 1 to 10 ng/mL of TGF-beta 1 resulted in a maintenance of high proliferative potential-colony-forming cell (HPP-CFC) during 8 days of culture compared with loss of HPP-CFC in cultures with no added TGF- beta 1. In conclusion, this study shows that TGF-beta 1 directly inhibits the initial stages of proliferation of LTR-HSC and appears to slow the differentiation of daughter cells of low Ho/Rh cells.     

Shp-2 heterozygous hematopoietic stem cells have deficient repopulating ability due to diminished self-renewal

OBJECTIVE: Improved understanding of hematopoietic stem cell (HSC) differentiation, proliferation, and self-renewal is sought to develop improved stem cell-based therapies as well as to define novel therapies for stem cell-based diseases such as leukemia. Shp-2 is a widely expressed nonreceptor protein tyrosine phosphatase that participates early in hematopoietic development. The following study was performed to examine the role of Shp-2 in HSC function. METHODS: Bone marrow low-density mononuclear cells were isolated from WT and Shp-2(+/-) littermate controls and utilized in competitive repopulation studies, homing analysis, cell-cycle analysis, and serial transplantation studies. RESULTS: Haploinsufficiency of Shp-2 causes a threefold reduction in HSC repopulating units following transplantation into lethally irradiated recipients. Homing of Shp-2(+/-) and WT cells to the bone marrow and spleen compartments was equal. Cell-cycle analysis studies revealed that the Shp-2(+/-) lin(-)Sca-1(+)c-kit(+) cells are less quiescent than WT cells, providing a potential etiology for the observed reduced engraftment of the Shp-2(+/-) cells. Consistently, in serial transplantation studies, we observed a significant reduction of Shp-2(+/-) self-renewal compared to that of WT cells. CONCLUSION: These data demonstrate that Shp-2 is required for the physiologic homeostasis of the HSC compartment and potentially provide insight into how oncogenic Shp-2 may contribute to the pathogenesis of myeloproliferative disorders and leukemias.     

Transplantation of a myelodysplastic syndrome by a long-term repopulating hematopoietic cell

The myelodysplastic syndromes (MDS) comprise a group of premalignant hematologic disorders characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). Although it is well established that many malignancies can be transplanted, there is little evidence to demonstrate that a premalignant disease entity, such as MDS or colonic polyps, can be transplanted and subsequently undergo malignant transformation in vivo. Using mice that express a NUP98-HOXD13 (NHD13) transgene in hematopoietic tissues, we show that a MDS can be transplanted to WT recipients. Recipients of the MDS bone marrow displayed all of the critical features of MDS, including peripheral blood cytopenias, dysplasia, and transformation to AML. Even when transplanted with a 10-fold excess of WT cells, the NHD13 cells outcompeted the WT cells over a 38-week period. Limiting-dilution experiments demonstrated that the frequency of the cell that could transmit the disease was ≈1/6,000–1/16,000 and that the MDS was also transferable to secondary recipients as a premalignant condition. Transformation to AML in primary transplant recipients was generally delayed (46–49 weeks after transplant); however, 6 of 10 secondary transplant recipients developed AML. These findings demonstrate that MDS originates in a transplantable, premalignant, long-term repopulating, MDS-initiating cell.     

Enforced expression of MLL-AF4 fusion in cord blood CD34+ cells enhances the hematopoietic repopulating cell function and clonogenic potential but is not sufficient to initiate leukemia

Infant acute lymphoblastic leukemia harboring the fusion mixed-lineage leukemia (MLL)-AF4 is associated with a dismal prognosis and very brief latency. Our limited understanding of transformation by MLL-AF4 is reflected in murine models, which do not accurately recapitulate the human disease. Human models for MLL-AF4 disease do not exist. Hematopoietic stem or progenitor cells (HSPCs) represent probable targets for transformation. Here, we explored in vitro and in vivo the impact of the enforced expression of MLL-AF4 in human cord blood-derived CD34(+) HSPCs. Intrabone marrow transplantation into NOD/SCID-IL2Rγ(-/-) mice revealed an enhanced multilineage hematopoietic engraftment, efficiency, and homing to other hematopoietic sites on enforced expression of MLL-AF4. Lentiviral transduction of MLL-AF4 into CD34(+) HSPCs increased the in vitro clonogenic potential of CD34(+) progenitors and promoted their proliferation. Consequently, cell cycle and apoptosis analyses suggest that MLL-AF4 conveys a selective proliferation coupled to a survival advantage, which correlates with changes in the expression of genes involved in apoptosis, sensing DNA damage and DNA repair. However, MLL-AF4 expression was insufficient to initiate leukemogenesis on its own, indicating that either additional hits (or reciprocal AF4-MLL product) may be required to initiate ALL or that cord blood-derived CD34(+) HSPCs are not the appropriate cellular target for MLL-AF4-mediated ALL.     

Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient "compromised" bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.     

Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil- treated murine marrow

Lymphomyeloid stem cells from the bone marrow of C57BL/6 mice treated with 5-fluorouracil (5-FU) were characterized with respect to 12 parameters using fluorescence-activated cell sorting and a competitive long-term repopulation assay. Stem cells were larger than lymphocytes and exhibited side light-scatter characteristic of blast cells. Most expressed low levels of Thy-1.2, high levels of Sca-1 (Ly6-A/E), H-2Kb, and AA4.1 antigens and stained brightly with rhodamine-123. Significantly, most long-term repopulating cells also expressed CD4, some at high density. In addition, a significant proportion displayed low to medium levels of the "lineage-specific" markers CD45R (B220), Gr- 1, and TER-119. A simple and rapid multiparameter sorting procedure enriched the stem cells 100-fold and substantially removed most other clonogenic cell types, including day 12 spleen colony-forming cells. Cells able to generate cobblestone colonies on stromal cells in vitro were co-enriched. Lethally irradiated mice transplanted with limiting numbers of the sorted stem cells did not survive unless cotransplanted with "compromised" marrow cells prepared by prior serial transplantation and shown to be depleted of long-term repopulating activity. A significant number of recipients transplanted with 25 to 100 sorted cells contained donor-derived B and T lymphocytes and granulocytes in their peripheral blood for at least 6 months. Limiting dilution analysis in vivo indicated that the frequency of competitive long-term repopulating units (CRU) in the sorted population was at least 1 in 60 cells. The calculated frequency of CRU was largely independent of the time of recipient analysis between 10 and 52 weeks, indicating that highly enriched stem cells can be recruited relatively early in certain transplant settings. This simple enrichment and assay strategy for repopulating hematopoietic stem cells should facilitate further analysis of their regulation in vivo.     

BMP signaling is required for postnatal murine hematopoietic stem cell self-renewal

Life-long production of blood from hematopoietic stem cells (HSC) is a process of strict modulation. Intrinsic and extrinsic signals govern fate options like self-renewal – a cardinal feature of HSC. Bone morphogenetic proteins (BMP) have an established role in embryonic hematopoiesis, but less is known about its functions in adulthood. Previously, SMAD-mediated BMP signaling has been proven dispensable for HSC. However, the BMP type-II receptor (BMPR-II) is highly expressed in HSC, leaving the possibility that BMP function via alternative pathways. Here, we establish that BMP signaling is required for selfrenewal of adult HSC. Through conditional knockout we show that BMPR-II deficient HSC have impaired self-renewal and regenerative capacity. BMPR-II deficient cells have reduced p38 activation, implying that non-SMAD pathways operate downstream of BMP in HSC. Indeed, a majority of primitive hematopoietic cells do not engage in SMADmediated responses downstream of BMP in vivo. Furthermore, deficiency of BMPR-II results in increased expression of TJP1, a known regulator of self-renewal in other stem cells, and knockdown of TJP1 in primitive hematopoietic cells partly rescues the BMPR-II null phenotype. This suggests TJP1 may be a universal stem cell regulator. In conclusion, BMP signaling, in part mediated through TJP1, is required endogenously by adult HSC to maintain self-renewal capacity and proper resilience of the hematopoietic system during regeneration.     

Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male "test" cells and 1 to 2 x 10(5) "compromised" female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5-FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.     

HLA matching enhances long-term renal graft survival but does not relate to acute rejection

Forty patients with end-stage chronic renal failure received living donor renal grafts, matched at more than 1 HLA haplotype, over the last 25 years. Of these grafts, 33 were first and 7 were second grafts. All recipients received prophylactic corticosteroids. Thirty-four also received prophylactic azathioprine, and 6, prophylactic cyclosporine. Acute rejection occurred in 65% (11/17) of non-cyclosporine treated grafts when the recipient was given 5 or fewer units of blood preoperatively, but in only 18% (3/17) when more than 5 units were given. High-dose steroid therapy reversed the acute rejection each time. Chronic rejection occurred in 2 grafts. Irreversible rejection did not occur in any second graft. Chronic glomerulonephritis, possibly due to recurrent disease, occurred in 1 graft. Five grafts have been lost, 1 each from technical and immunosuppressive complications, and 3 from incidental death. The 1-year actuarial graft survival rate is 95% and the 10-year rate 84%. All surviving patients lead normal lives without significant health restriction, and employ minimal medication. It is postulated that: 1) acute cellular rejection is HLA-independent, and chronic rejection is HLA-dependent, and 2) hyperacute and chronic rejection are related and are parts of a spectrum of humoral immunity.     

Cultivation of aorta-gonad-mesonephros-derived hematopoietic stem cells in the fetal liver microenvironment amplifies long-term repopulating activity and enhances engraftment to the bone marrow

During mammalian development, definitive hematopoietic stem cells (HSCs) arise in the aorta-gonad-mesonephros (AGM) region and colonize the fetal liver (FL) before hematopoiesis occurs in the bone marrow. The FL is a unique hematopoietic organ where both HSCs and mature blood cells are actively generated along with functional maturation of hepatic cells as a metabolic organ. To characterize HSCs and FL microenvironments during development, this study establishes a coculture system composed of AGM-originated HSCs and FL nonhematopoietic cells. The results demonstrate that FL cells support significant expansion of lineage-committed hematopoietic cells as well as immature progenitors. More important, long-term repopulating activity was amplified from AGM-originated HSCs in this coculture system. Engraftment of HSCs to the bone marrow was strongly enhanced by coculture. In addition, AGM HSCs produced significantly more hematopoietic cells than E14.5 and E18.5 FL HSCs in vitro. These results suggest that the FL microenvironment not only stimulates expansion of the hematopoietic system, but also possibly modifies the characteristics of AGM HSCs. Thus, this coculture system recapitulates the developmental process of HSCs and the FL microenvironment and provides a novel means to study the development of hematopoiesis.     

An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse

We have developed a limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse using a miniturized stroma- dependent bone marrow culture assay in vitro. The cells were overlaid on irradiated stromal layers in microtiter wells in a range of concentrations, and frequencies of cobblestone area-forming cells (CAFC) were calculated by employing Poisson statistics. The production of secondary granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer of individual wells was correlated with the presence of such cobblestone areas. CAFC frequencies were determined in bone marrow cell suspensions that were either enriched for marrow repopulating ability (MRA) in vivo, while depleted for spleen colony- forming units (CFU-S), or vice versa. The separation of bone marrow cells (BMC) was either based on centrifugal elutriation, or monoclonal antibody-mediated magnetic depletion of cells carrying cell surface differentiation antigens, and subsequent sorting on the basis of light scatter and rhodamine-123 retention as a measure of mitochondrial activity. In addition, 5-fluorouracil-resistant BMC were studied. Our investigations show that a time-dependent cobblestone area formation exists that reflects the turnover time and primitiveness of CAFC. The frequency of precursors forming cobblestone areas on day 28 after overlay is proposed to be a measure for MRA, whereas the day-7 CAFC frequency closely corresponds with day-12 CFU-S numbers in the suspensions tested.     

Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture.

Recently, culture conditions that stimulate the proliferation of primitive hematopoietic cells defined by various phenotypic and functional endpoints in vitro have been identified. However, evidence that they support a high probability of self-renewal leading to a large net expansion in vitro of transplantable cells with lympho-myeloid repopulating ability has been more difficult to obtain. The present study was designed to investigate whether the low overall expansion of human repopulating hematopoietic cells seen in vitro reflects a selective unresponsiveness of these rare cells to the growth factors currently used to stimulate them or, alternatively, whether they do proliferate in vitro but lose engrafting potential. For this, we used a high-resolution procedure for tracking and reisolating cells as a function of their proliferation history based on the loss of cellular fluorescence after staining with (5- and 6-) carboxyfluorescein diacetate succinimidyl ester. The results show that the vast majority of long-term culture-initiating cells and in vivo lympho-myeloid competitive repopulating units present in 5-day suspension cultures initiated with CD34(+) human cord blood and fetal liver cells are the progeny of cells that have divided at least once in response to stimulation by interleukin-3, interleukin-6, granulocyte colony-stimulating factor, Steel factor, and Flt3-ligand. Thus, most human repopulating cells from these two sources are stimulated to undergo multiple divisions under currently used short-term suspension culture conditions and a proportion of these retain engraftment potential.