A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38- progenitor cells in long-term cultures and semisolid assays

Analysis of molecular mechanisms associated with stem cell commitment and differentiation requires an in vitro assay that identifies the most primitive hematopoietic stem cells in human bone marrow. Such primitive stem cells usually do not form colonies in short-term semisolid assays and are best identified by their ability to initiate sustained hematopoiesis when they are cocultured with competent stromal cells. In this study, we investigated whether a murine marrow stromal cell line (MS-5) that supports colony-forming unit-spleen (CFU-S) maintenance would permit, both in short-term colony assays and long-term cultures, the development of primitive human stem cells sorted on the basis of their high expression of CD34 and lack of expression of CD38 antigen. In short-term colony assays, this population included almost exclusively primitive progenitor cells. MS-5 cells synergized with any combination of interleukin-3, Steel factor, granulocyte colony- stimulating factor, agar-leukocyte conditioned medium, and erythropoietin and increased at least twofold both the cloning efficiency of CD34++/CD38- cells and the size of the colonies. Furthermore, MS-5 cells triggered the development of multipotent blast cell progenitors with a high proliferative potential, which in these conditions represented 1% to 2% of CD34++/CD38- cells. When MS-5 cells were substituted by human stromal cells or when growth factor combinations were used in the absence of stromal cells, much lower numbers of CFU-blast were detected. This selective action of MS-5 on early progenitors was also observed when MS-5 cells were used as feeders in long-term cultures of CD34++/CD38- cells. Murine cells promoted the expansion of high proliferative potential primitive progenitor cells up to 3 months, although they did not support their differentiation in mature clonogenic progenitors or terminally differentiated cells. Sustained hematopoiesis in these longterm cultures was accounted for by 2% to 5% of initial CD34++/CD38- cells as estimated by limiting dilution experiments. Mechanisms by which murine stromal cells act specifically on human primitive stem cells are unclear, but from our data this effect is unlikely to be explained solely by known species cross-reactive growth factors. Further manipulation of this long-term coculture system should prove useful in identifying stromal molecules regulating commitment and differentiation of early human progenitor cells.     

Expression and function of CD105 during the onset of hematopoiesis from Flk1(+) precursors.

During ontogeny, the hematopoietic system is established from mesoderm-derived precursors; however, molecular events regulating the onset of hematopoiesis are not well characterized. Several members of the transforming growth factor beta (TGF-beta) superfamily have been implicated as playing a role during mesoderm specification and hematopoiesis. CD105 (endoglin) is an accessory receptor for members of the TGF-beta superfamily. Here it is reported that during the differentiation of murine embryonic stem (ES) cells in vitro, hematopoietic commitment within Flk1(+) mesodermal precursor populations is characterized by CD105 expression. In particular, CD105 is expressed during the progression from the Flk1(+)CD45(-) to Flk1(-)CD45(+) stage. The developmentally regulated expression of CD105 suggests that it may play a role during early hematopoiesis from Flk1(+) precursors. To determine whether CD105 plays a functional role during early hematopoietic development, the potential of CD105-deficient ES cells to differentiate into various hematopoietic lineages in vitro was assessed. In the absence of CD105, myelopoiesis and definitive erythropoiesis were severely impaired. In contrast, lymphopoiesis appeared to be only mildly affected. Thus, these findings suggest that the regulated expression of CD105 functions to support lineage-specific hematopoietic development from Flk1(+) precursors.     

Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells

The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDH(hi)Lin- cells). Here, we further dissected the ALDH(hi)-Lin- population by selection for CD133, a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. ALDH(hi)CD133+Lin- cells were primarily CD34+, but also included CD34-CD38-CD133+ cells, a phenotype previously associated with repopulating function. Both ALDH(hi)CD133-Lin- and ALDH(hi)CD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro, whereas only the ALDH(hi)CD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID beta2M-null mice that received transplants of ALDH(hi)CD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells, suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDH(hi)CD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial, secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies.     

Hematopoiesis and stem cell renewal in long-term bone marrow cultures containing catalase

Culturing mouse bone marrow in the presence of catalase dramatically alters hematopoiesis. Granulocyte output is initially increased 4- to 5-fold. This increase is transient and granulocyte production declines as immature (Sca-1+/LIN-) cells accumulate. One third of these immature cells have a phenotype (Sca-1+/c-Kit+) characteristic of hematopoietic stem cells. At 2 to 3 weeks there are greater than 200-fold more Sca-1+/c-Kit+/LIN- cells in treated cultures than in controls. This population contains functional stem cells with both short-term and long-term bone marrow repopulating activity. In addition to myeloid progenitors, this Sca-1+/LIN- population contains a large number of cells that express CD31 and CD34 and have an active Tie-2 promoter, indicating that they are in the endothelial lineage. After 3 to 4 weeks hematopoiesis in treated cultures wanes but if catalase is removed, hematopoiesis resumes. After 7 to 10 days the cultures are indistinguishable from untreated controls. Thus, protected from H2O2, hematopoietic progenitors multiply and become quiescent. This sequence resembles in vivo development in normal marrow. These results make it clear that peroxide-sensitive regulatory mechanisms play an important role in controlling hematopoiesis ex vivo and presumably in vivo as well. They also indicate that manipulation of the peroxide levels can be used to enhance the growth of hematopoietic stem cells in culture.     

Use of CD9 expression to enrich for porcine hematopoietic progenitors

OBJECTIVE: The aim of this study was to develop novel markers for enrichment of hematopoietic progenitors from bone marrow of swine. MATERIALS AND METHODS: We previously showed that pig bone marrow contains a "side population" (SP) of Hoechst dye-effluxing cells that resembles the hematopoietic stem cell (HSC)-containing murine SP and therefore represents a putative pig stem cell population. We screened a panel of monoclonal antibodies for those that allowed positive or negative enrichment of porcine SP cells and tested one of these for enrichment of hematopoietic progenitors in short-term and long-term in vitro assays. We then screened an expression library to clone the gene whose product is recognized by this antibody. RESULTS: Among a panel of 35 monoclonal lines screened, we found three that were useful for positive enrichment of SP cells and seven for negative enrichment. The 4-6 monoclonal line, allowing around 10-fold negative enrichment of SP cells, recognized the product of the porcine CD9 gene. Hematopoietic progenitors measured by short-term colony-forming unit and long-term cobblestone area-forming cell assays were around 10-fold enriched in the CD9(negative/low) fraction and were significantly depleted in the CD9(high) fraction. CONCLUSIONS: The antibody against the porcine CD9 gene product may be of use for enrichment of porcine hematopoietic stem cells. This approach to identify novel markers for enrichment of hematopoietic progenitors may be applicable to other mammalian species.     

Expression of CD4 by human hematopoietic progenitors [see comments]

It has been recently reported that murine hematopoietic stem cells and progenitors express low levels of CD4. In this study, we have investigated by phenotypic and functional analysis whether the CD4 molecule was also present on human hematopoietic progenitors. Unfractionated marrow cells or immunomagnetic bead-purified CD34+ cells were analyzed by two-color fluorescence with an anti-CD4 and an anti- CD34 monoclonal antibody (MoAb). A large fraction (25% to 50%) of the CD34+ cells was weakly stained by anti-CD4 antibodies. Moreover, in further experiments analyzing the expression of CD4 in different subpopulations of CD34+ cells, we found that CD4 was predominantly expressed in phenotypically primitive cells (CD34+ CD38-/low CD71low Thy-1high, HLA-DR+/low). However, the presence of CD4 was not restricted to these primitive CD34+ cell subsets and was also detected in a smaller fraction of more mature CD34+ cells exhibiting differentiation markers. Among those, subsets with myelo-monocytic markers (CD13, CD33, CD14, and CD11b) have a higher CD4 expression than the erythroid or megakaryocytic subsets. In vitro functional analysis of the sorted CD34+ subsets in colony assays and long-term culture- initiating cell (LTC-IC) assays confirmed that clonogenic progenitors (colony-forming unit-granulocyte-macrophage, burst-forming unit- erythroid, and colony-forming unit-megakaryocyte) and LTC-IC were present in the CD4low population. However, most clonogenic progenitors were recovered in the CD4- subset, whereas the CD4low fraction was greatly enriched in LTC-IC. In addition, CD4low LTC-IC generated larger numbers of primitive clonogenic progenitors than did CD4- LTC-IC. These observations suggest that, in the progenitor compartment, the CD4 molecule is predominantly expressed on very early cells. The CD4 molecule present on CD34+ cells appeared identical to the T-cell molecule because it was recognized by three MoAbs recognizing different epitopes of the molecule. Furthermore, this CD4 molecule is also functional because the CD34+ CD4low cells are able to bind the human immunodeficiency virus (HIV) gp120. This observation might be relevant to the understanding of the mechanisms of HIV-induced cytopenias.     

PECAM-1 is expressed on hematopoietic stem cells throughout ontogeny and identifies a population of erythroid progenitors

Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31) is an adhesion molecule expressed on endothelial cells and subsets of leukocytes. Analysis of phenotypically defined hematopoietic stem cells (HSCs) from the yolk sac, fetal liver, and adult bone marrow demonstrates CD31 expression on these cells throughout development. CD31+ c-kit+ cells, but not CD31- c-kit+ cells, isolated from day-9.5 yolk sac give rise to multilineage hematopoiesis in vivo. Further evaluation of the CD31+ lineage marker-negative fraction of adult bone marrow reveals functionally distinct cell subsets. Transplantation of CD31+ Lin- c-kit- cells fails to protect lethally irradiated recipients, while CD31+ Lin- c-kit+ Sca-1- cells (CD31+ Sca-1-) provide radioprotection in the absence of long-term donor-derived hematopoiesis. Although donor-derived leukocytes were not detected in CD31+ Sca-1- recipients, donor-derived erythroid cells were transiently produced during the initial phases of bone marrow recovery. These results demonstrate CD31 expression on hematopoietic stem cells throughout ontogeny and identify a population of CD31+ short-term erythroid progenitors cells that confer protection from lethal doses of radiation.     

Vascular cell adhesion molecule-1 expressed by bone marrow stromal cells mediates the binding of hematopoietic progenitor cells.

Human bone marrow-derived CD34+ cells were analyzed for the expression of the beta 1-family of integrin adhesion molecules. Integrin alpha 4 beta 1 was consistently expressed by greater than 90% of CD34+ cells, including essentially all assayable granulocyte-macrophage colony-forming cells (CFU-GM) and erythroid bursts (BFU-E) as shown by fluorescence-activated cell sorting studies. Adhesion of highly enriched CD34+ cells to cultured allogeneic marrow stromal cells was largely inhibited both by monoclonal antibody to alpha 4 beta 1 and to vascular cell adhesion molecule-1 (VCAM-1), a ligand for alpha 4 beta 1. VCAM-1 was found to be expressed by bone marrow stromal elements in vitro both constitutively at low level and at high levels after treatment with cytokines. Induction of VCAM-1 was cytokine- and time-dependent with maximum levels being obtained after 4 hours of exposure to a combination of interleukin-4 and tumor necrosis factor-alpha. Cytokine-induced stromal cells bound threefold higher numbers of CFU-GM and BFU-E, this increase being abrogated by anti-alpha 4 beta 1 and anti-VCAM-1 antibodies. In addition, the adhesion to stroma of more immature progenitors, the long-term culture initiating cells, also occurred through an alpha 4 beta 1/VCAM-1-dependent mechanism. These studies identify an adhesion mechanism of potential importance in the localization of primitive progenitors within the hematopoietic microenvironment.     

Expansion of multipotent and lymphoid-committed human progenitors through intracellular dimerization of Mpl

Self-renewal capacity is rapidly lost during differentiation of hematopoietic stem cells to lineage-committed progenitors. We demonstrate here that regulated intracellular signaling through the cytokine receptor Mpl induces profound expansion of not only multipotent (ie, lymphomyeloid) but also lymphoid-committed human hematopoietic progenitors. A fusion protein containing the intracellular signaling domain of Mpl and a dimerization domain was constitutively expressed in populations enriched in human lymphomyeloid progenitor/stem cells (CD34(+)CD38(-)Lin(-)CD7(-)) and multilymphoid progenitors (CD34(+)CD38(-)Lin(-)CD7(+)). Intracellular dimerization of Mpl in target cells was induced by in vitro or in vivo administration of a diffusible synthetic ligand. In vitro, Mpl dimerization produced divisions of clonogenic, multilineage CD34(+) cells able to engraft immunodeficient mice. When dimerization was induced in vivo after transplantation of either lymphomyeloid or multilymphoid progenitors, donor-derived hematopoiesis was sustained for at least 12 weeks and primitive CD34(+)Lin(-) progenitors were expanded more than 1000-fold. Lineage potential of progenitors was not altered and differentiation was not prevented by synthetically induced Mpl signaling. These data demonstrate that dimerization of a single cytokine receptor can deliver a profound expansion signal in both uncommitted and lymphoid-committed human hematopoietic progenitors.     

Characterization of murine CD34, a marker for hematopoietic progenitor and stem cells

CD34 is expressed on human hematopoietic stem and progenitor cells, and its clinical usefulness for the purification of stem cells has been well established. However, a similar pattern of expression for murine CD34 (mCD34) has not yet been determined. Two polyclonal anti-mCD34 antibodies that specifically recognize both endogenous and recombinant murine CD34 were developed to characterize the mCD34 protein and to determine its pattern of expression on murine cell lines and hematopoietic progenitor cells. Fluorescence-activated cell sorter analysis showed that mCD34 is expressed on NIH/3T3 embryonic fibroblasts, PA6 stromal cells, embryonic stem cells, M1 leukemia cells, and a subpopulation of normal bone marrow cells. Murine CD34 was found to be a glycoprotein expressed on the cell surface as either a full-length (approximately 100 kD) or truncated (approximately 90 kD) protein in NIH/3T3 and PA6 cells. Recombinant full-length CD34, when expressed in the CHO-K1 cell line, had a molecular weight of approximately 105 kD. Full-length CD34 expressed on M1 leukemia cells, had a higher apparent molecular weight (110 kD). These results suggest that there are glycosylation differences between CD34 expressed by different cell types. The full-length form, but not the truncated form, is a phosphoprotein that is hyperphosphorylated in response to 12-0- Tetradecanoyl phorbol 13-acetate treatment, suggesting potential functional differences between the two forms. Selection of the 3% highest-expressing CD34+ bone marrow cells enriched for the hematopoietic precursors that form colony-forming unit-spleen (CFU-S), CFU-granulocyte-macrophage, and burst-forming unit-erythroid. Transplantation of lethally irradiated mice with these cells demonstrated both short- and long-term repopulating ability, indicating that this population contains both functional hematopoietic progenitors and the putative stem cell. These antibodies should be useful to select for murine hematopoietic stem cells.     

Stromal cell CD9 regulates differentiation of hematopoietic stem/progenitor cells

CD9 belongs to the transmembrane 4 superfamily, and has been shown to influence cell proliferation, motility, and adhesion. We show here that ligation of CD9 modifies proliferation and/or differentiation of hematopoietic stem/progenitors. Pluripotent EML-C1 hematopoietic cells were cocultured with MS-5 stromal cells in the presence of KMC8.8, an anti-CD9 antibody. Numbers of recovered EML-C1 cells were slightly reduced and the antibody caused the hematopoietic cells to migrate beneath the adherent stromal cell layer. Of particular interest, EML-C1 cells recovered from CD9-ligated cultures had undifferentiated properties. Separate pretreatment of the two cell types with antibody showed that stromal-cell CD9 mediated these responses. Spontaneous expression of erythroid marker was completely blocked and there was a shift towards undifferentiated clonogenic progenitors. Immunoprecipitation studies showed that stromal-cell CD9 associates with the beta1 subunit of integrin, as well as a novel 100 kD protein. Antibody cross-linking of cell surface CD9 increased the amount of 100 kD protein that was subsequently coprecipitated with CD9. These observations show that stromal-cell CD9 influences physical interactions with hematopoietic cells and may be one factor that determines the degree of stem cell differentiation.     

Transforming growth factor beta directly regulates primitive murine hematopoietic cell proliferation

We previously reported that transforming growth factor beta (TGF-beta) selectively inhibits colony-stimulating factor-driven hematopoietic progenitor cell growth. We report here that TGF-beta 1 can act directly on hematopoietic progenitors to inhibit the growth of the most primitive progenitors measurable in vitro. Highly enriched populations of hematopoietic progenitor cells were obtained by isolating lineage negative (Lin-), Thy-1-positive (Thy-1+) fresh bone marrow cells, or by isolating cells from interleukin-3 (IL-3) supplemented bone marrow cultures expressing Thy-1 antigen with the fluorescent activated cell sorter. TGF-beta 1 inhibited IL-3-induced Thy-1 expression on Thy-1-negative (Thy-1-) bone marrow cells in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. In addition, TGF-beta 1 inhibited the formation of multipotent and mixed colonies by isolated Thy-1+ cells, while single lineage granulocyte and macrophage colonies were not affected. The growth of Thy-1+ Lin- cells incubated as single cells in Terasaki plates in medium supplemented with IL-3 were inhibited by TGF-beta, demonstrating a direct inhibitory effect. Hematopoietic stem cells, which have a high proliferative potential (HPP) when responding to combinations of growth factors in vitro, have been detected in the bone marrow of normal mice and mice surviving a single injection of 5-fluorouracil. TGF-beta 1 inhibited the growth of all subpopulations of HPP colony forming cells (CFC) in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. Thus, TGF-beta directly inhibits the growth of the most immature hematopoietic cells measurable in vitro.     

Primitive hematopoietic cells in murine bone marrow express the CD34 antigen

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin- /10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.     

Isolation of a candidate human hematopoietic stem-cell population.

We have identified a rare (0.05-0.1%) subset of human fetal bone marrow cells that contains multipotent hematopoietic precursors. The population of human precursor cells that express Thy-1 and CD34 but no known lineage markers is enriched for clonogenic activity that establishes long-term, multilineage (myelomonocytic and B lymphoid) cultures on mouse marrow stromal lines. Further, the Thy-1+CD34+ subset that takes up little of the fluorescent mitochondrial dye rhodamine 123 contains virtually all the cells that establish long-term cultures. In human fetal thymus transplanted into SCID (severe combined immunodeficiency) mice, Thy-1+CD34+ fetal bone marrow cells differentiate into T lymphocytes. In two of nine cases, allogeneic Thy-1+CD34+ cells could engraft intact human fetal bone marrow grown in SCID mice, resulting in donor-derived myeloid and B cells. By extrapolation, the rare human Thy-1+Lin-CD34+ cell population contains pluripotent hematopoietic progenitors; we propose that it is highly enriched for candidate hematopoietic stem cells.     

Regulation of hematopoiesis in vitro and in vivo by invariant NKT cells

Invariant natural killer T cells (iNKT cells) are a small subset of immunoregulatory T cells highly conserved in humans and mice. On activation by glycolipids presented by the MHC-like molecule CD1d, iNKT cells promptly secrete T helper 1 and 2 (Th1/2) cytokines but also cytokines with hematopoietic potential such as GM-CSF. Here, we show that the myeloid clonogenic potential of human hematopoietic progenitors is increased in the presence of glycolipid-activated, GM-CSF-secreting NKT cells; conversely, short- and long-term progenitor activity is decreased in the absence of NKT cells, implying regulation of hematopoiesis in both the presence and the absence of immune activation. In accordance with these findings, iNKT-cell-deficient mice display impaired hematopoiesis characterized by peripheral-blood cytopenias, reduced marrow cellularity, lower frequency of hematopoietic stem cells (HSCs), and reduced early and late hematopoietic progenitors. We also show that CD1d is expressed on human HSCs. CD1d-expressing HSCs display short- and long-term clonogenic potential and can present the glycolipid alpha-galactosylceramide to iNKT cells. Thus, iNKT cells emerge as the first subset of regulatory T cells that are required for effective hematopoiesis in both steady-state conditions and under conditions of immune activation.