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PubMed1990

Human adenosine deaminase expression in mice.

A replication defective retroviral vector containing a human adenosine deaminase (hADA) cDNA was produced by GP + E-86 packaging cells at high titer. We report long-term expression of hADA in the hematopoietic tissues of mice transplanted with bone marrow cells infected by in vitro co-cultivation with vector producing cells. Western analysis using an hADA-specific antibody allowed detection of the protein in the peripheral blood of all 37 transplanted mice for at least 9 weeks. Sixty-eight percent of the animals continued to express hADA in one or more of their hematopoietic tissues for the experimental period, and hADA was found in both spleen colonies and tissues of secondary recipients. There was provirus integration and expression in myeloid, erythroid, and lymphoid cell lineages, indicating extensive repopulation by the progeny of infected stem cells. The vector did not contain a selectable marker, and the infected stem cells did not have a competitive in vivo advantage. Nevertheless, we observed consistent gene transfer into hematopoietic stem cells and long-term expression of a human gene product in their progeny.

Adenosine Deaminase↗
PubMed2005

c-fos overexpression in splenic B cells augments development of marginal zone B cells.

Marginal zone (MZ)-B cells participate in very early immune responses and play a pivotal role in the first-line of defense against blood-borne Ags including bacterial LPS. Since splenic B cells from c-fos transgenic (H2-c-fos) mice are hyper-sensitive to LPS stimulation, we examined LPS-sensitivity of MZ-B cells in the spleen of H2-c-fos mice. Here, we show that proliferation of MZ-B cells from H2-c-fos mice stimulated with LPS was larger than that from control mice. Proliferation and IgM production of the H2-c-fos MZ-B cells were also larger than those of splenic follicular (FO)-B cells from the H2-c-fos mice, suggesting that c-fos overexpression augments LPS-sensitivity of MZ-B cells more than that of FO-B cells. Furthermore, the number of MZ-B cells but not that of FO-B cells in the spleen of H2-c-fos mice was two- to three-fold larger than that in control littermates. The number of transitional type II (T2)-B cells in H2-c-fos mice was also larger than that of control littermates. However, the number of transitional type I (T1)-B cells in the spleen of H2-c-fos mice was not larger than that of control mice. Moreover, this c-fos effect on differentiation of MZ-B cells was intrinsic in B cells by the competitive repopulation assay with hematopoietic stem cells of H2-c-fos and control mice. These results suggest that c-fos overexpression in B cells augments differentiation and accumulation of MZ-B cells.

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PubMed1998

Stem cell factor improves the repopulating ability of primitive hematopoietic stem cells after sublethal irradiation (and, to a lesser extent) after bone marrow transplantation in mice.

Bone marrow transplantation (BMT) and sublethal irradiation (XRT) cause profound long-term damage to hematopoietic stem cells. We used the competitive repopulation assay in mice to test the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF), cytokines given in clinical settings to enhance marrow recovery after XRT or BMT and to protect the marrow repopulating ability of primitive hematopoietic stem cells (PHSC) after these modalities. The repopulating ability of exhaustible multilineage progenitors (EMP) was also tested after these modalities, with or without cytokines. Repopulating abilities of EMP and PHSC were significantly reduced after XRT or BMT; PHSC were preferentially affected. Administration of SCF to C57B6/J mice after XRT resulted in improved EMP and PHSC repopulating ability, although progenitor numbers--repopulating units--were not completely returned to control levels. Whether given as a single dose or multiple doses, GM after XRT did protect PHSC function from the deleterious effects of XRT, but this was not a significant effect. SCF caused an increase in PHSC repopulating ability after BMT, but this too was not a significant difference. GM after BMT had little effect. SCF administration before XRT led to severe impairment of PHSC function with very little or no stem cell activity observed. Therefore, timing of its administration is an important consideration since preadministration of the cytokine before XRT can be extremely harmful to PHSC function.

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PubMed1999

Functional activity of murine CD34+ and CD34- hematopoietic stem cell populations.

The transmembrane glycoprotein CD34 is expressed on human hematopoietic stem cells and committed progenitors in the bone marrow, and CD34-positive selection currently is used to isolate bone marrow repopulating cells in clinical transplantation protocols. Recently, CD34- hematopoietic stem cells were described in both humans and mice, and it was suggested that CD34+ murine bone marrow cells may lack long-term reconstituting ability. In this study, the long-term repopulating ability of CD34+Lin- vs CD34-Lin- cells was compared directly using syngeneic murine bone marrow transplantation. Highly purified populations of CD34+Lin- and CD34-Lin- cells each are able to reconstitute bone marrow, confirming that both populations contain hematopoietic stem cells; however, the number of hematopoietic stem cells in the CD34+Lin- fraction is approximately 100-fold greater than the number in the CD34-Lin- fraction. In competitive repopulation experiments, CD34+ stem cells are better able to engraft the bone marrow than are CD34- cells. CD34+Lin- cells provide both short- and long-term engraftment, but the CD34-Lin- cells are capable of only long-term engraftment. Ex vivo, the CD34+Lin- stem cells expand over 3 days in culture and maintain the ability to durably engraft animals in a serial transplant model. In contrast, when CD34-Lin- cells are cultured using the same conditions ex vivo, the cell number decreases, and the cells do not retain the ability to repopulate the bone marrow. Thus, the CD34+Lin- and CD34-Lin- cells constitute two functionally distinct populations that are capable of long-term bone marrow reconstitution.

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PubMed1998

Lymphohematopoietic engraftment in minimally myeloablated hosts.

The concept that myeloablation to open space was a prerequisite for marrow stem cell engraftment has been challenged by studies showing high rates of engraftment in nonmyeloablated mice (Stewart et al, Blood 81:2566, 1993; Quesenberry et al, Blood Cells 20:97, 1994; Brecher et al, Blood Cells 5:237, 1979; Saxe et al, Exp Hematol 12:277, 1984; and Wu et al, Exp Hematol 21:251, 1993). However, relatively large numbers of marrow cells were necessary to achieve high long-term donor percentages. We have demonstrated, using a BALB/c male/female marrow transplant model and detecting male DNA in host tissues by Southern blot or fluorescent in situ hybridization, that exposure to doses of irradiation that cause minimal myeloablation (50 to 100 cGy) leads to very high levels of donor chimerism, such that relatively small numbers of marrow cells (10 to 40 million) can give donor chimerism in the 40% to 100% range. Studies of radiation sensitivity of long-term engrafting cells have shown that 100 cGy, although not myelotoxic, is stem cell toxic, and indicate that the final host:donor ratios are determined by competition between host and donor stem cells. These data indicate that low levels of irradiation should be an effective approach to nontoxic marrow transplantation in gene therapy or in attempts to create allochimerism to treat such diseases as cancer, sickle cell anemia, or thalassemia.

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PubMed1996

A comparison of long-term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse.

Previous studies have shown that stem cells able to competitively reconstitute the hematopoietic system of lethally irradiated mice (competitive repopulating units [CRU]) can be obtained in highly purified form from adult mouse bone marrow (BM) by the isolation of cells with a Sca-1+Lin-WGA+ phenotype. We now report on the phenotypic characteristics of CRU from day-14.5 murine fetal liver (FL). Our results confirm previous reports of similarities between the two CRU populations but also reveal a few striking differences. Both were found to express the Sca-1 antigen (SCA-1+ and surface molecules that bind wheat germ agglutinin (WGA+), and both show an absence or low expression of a number of markers characteristic of mature hematopoietic cells: B220, Gr-1,ly-1 and Ter119 (together termed Lin*-). Limiting dilution analysis of recipients transplanted with purified Sca-1+Lin*- FL cells with intermediate forward- and side-scatter properties showed that the frequency of CRU in this FL subpopulation was one in 39 cells. This represents an enrichment of approximately 450-fold over the labeled but unseparated FL starting population (one in 17,300 total FL cells). These FL CRU also resembled their counterparts in adult BM in that they expressed high levels of MHC class I and CD43 and intermediate levels of heat-stable antigen (HSA) and c-kit and did not express, or expressed at a low level, Thy-1.2, CD71, and the antigen recognized by the Fall-3 monoclonal antibody (mAb). In contrast, a high percentage of the Sca-1+Lin*- cells isolated from 14.5-day-old FL stained with the AA4.1, anti-Mac-1, and the anti-CD45RB mAbs and retained Rhodamine 123 (Rh123(bright)), whereas the Sca-1+Lin-WGA+ CRU-containing fraction of adult BM cells was found to be AA4.1-, Mac-1-, CD45RB-, and Rh123(dull). These differences in phenotype between CRU in FL and adult BM indicate changes that occur during ontogeny in cells that are similar with respect to their totipotentiality and long-term repopulating potential and complement parallel observations of functional differences between these two populations of CRU.

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PubMed1997

Engraftment of hematopoietic stem cells in nonmyeloablated and myeloablated hosts.

Hematopoietic stem cell engraftment has traditionally been assessed in irradiated murine hosts. More recently, we and others have shown that engraftment is virtually quantitative in host animals who have received no preconditioning myeloablation. It appears that at the stem cell level, engraftment may even be favored in the normal host. The final phenotypic readout in the engrafted animal is then determined by competition between the engrafted stem cells and the number of residual host stem cells. Further studies have indicated that with cytokine exposure in vitro, in vivo or 5-fluorouracil exposure and consequent stimulation of primitive hematopoietic stem cells to enter cell cycle, an engraftment defect relating to long-term engraftment occurs. This occurs in the face of an expansion of cycling surrogate stem cells as mirrored by the high proliferative potential colony-forming cell. These data indicate that the phenotype of the hematopoietic stem cell, as assessed in vitro, may not give insight into the phenotype of these cells in vivo.

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PubMed1988

Observations on the contributions of environmental restraints and innate stem cell ability to hematopoietic regeneration.

A competitive repopulation assay utilizing chromosome markers was used to assay the reconstituting potential of hematopoietic populations. The test populations consisted of tibial murine marrow locally irradiated with doses ranging from 1.5 Gy to 8.5 Gy and of marrow generated from either murine splenic or marrow stem cells. The purpose of this assay was to assess the innate proliferative potential and microenvironmental influences on the ability to repopulate. Regardless of origin, spleen repopulating ability consistently agreed with spleen colony-forming unit (CFU-s) content. Doses of radiation from 5 Gy to 8.5 Gy diminished, by a factor of 2, the ability to repopulate marrow despite maintenance of CFU-s levels. Marrow generated from splenic stem cells had one-fifth the repopulating ability of marrow derived from marrow stem cells, even though CFU-s levels were equivalent. The results imply that the splenic environment can only maintain stem cells at the level of the CFU-s, even if the stem cells were originally of higher quality, and that their original potential cannot be regained in a marrow environment. Nevertheless, the marrow can maintain more primitive stem cells, but this reserve is drained to support CFU-s levels.

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PubMed1991

Effects of hematopoietic growth factors on the survival of primitive stem cells in liquid suspension culture.

We have examined the effects of 10 different growth factors either alone or in combination on colony-forming unit-spleen (CFU-S) and repopulating stem cell survival in vitro. Either interleukin-3 (IL-3), granulocyte-colony-stimulating factor (G-CSF), or IL-4 alone support CFU-S in vitro. The effects of IL-3 or G-CSF could be neutralized by adding antibodies against IL-3 or G-CSF, respectively. However, the effects of IL-4 could be neutralized with antibodies to IL-4 as well as with antibodies to IL-3 and G-CSF. The combinations of IL-3 and IL-6, IL-3 and G-CSF, IL-3 and IL-1 alpha, IL-3 and granulocyte-macrophage CSF (GM-CSF), and IL-4 and IL-6 acted synergistically to increase CFU-S number. Addition of macrophage inflammatory protein-1 alpha (MIP-1 alpha) to IL-3 and IL-6 inhibited the increase in CFU-S number. Repopulating stem cell function was measured in a competitive repopulation assay. Either IL-3 or IL-4 alone could preserve stem cell function in vitro. The combinations of IL-3 and IL-6, and IL-3 and G-CSF increased stem cell function approximately twofold. The combinations of IL-3 + G-CSF + IL-6, and IL-4 and IL-6 (both of which increased CFU-S number fivefold to 10-fold) decreased stem cell function approximately fourfold. These results demonstrate that certain combinations of growth factors can increase CFU-S number at the expense of stem cell function.

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PubMed1992

Proliferation of totipotent hematopoietic stem cells in vitro with retention of long-term competitive in vivo reconstituting ability.

Marrow cells from male mice pretreated with 5-fluorouracil were infected with helper-free neomycin-resistant (neor) recombinant retrovirus and then used to initiate long-term cultures (LTC) on irradiated adherent marrow feeder layers. Four weeks later LTC cells were harvested and injected into lethally irradiated female recipients either alone or together with 2 x 10(5) female marrow cells with selectively compromised long-term repopulating potential to assay for totipotent and competitive repopulating units (CRU), respectively. A total of 46 unique clones were detected in recipients 5 wk to 7 mo after transplant. Half of these clones (22 of 46) included both lymphoid and myeloid progeny. Eight of the 22 lympho-myeloid clones were represented in multiple recipients, in some cases after injection of limiting numbers of CRU, thus indicating repopulation from sibling totipotent stem cells generated during the initial 4-wk period in LTC. Serial analysis of cells released into the nonadherent fraction of LTC for up to 7 wk provided additional evidence of the continuing proliferation in LTC of totipotent stem cells with long-term repopulating potential. The frequency of CRU determined from limiting-dilution analyses of LTC-derived cells was the same for recipients analyzed at 5 wk or 7 mo after transplantation and was also the same whether marrow or thymus repopulation was assessed. These assays showed that concurrent with the expansion of some totipotent cells revealed by retroviral marking, there was a slow but net 6.5-fold decrease in total CRU numbers after 4 wk in LTC. These results show the capacity of some totipotent hematopoietic stem cells to be maintained and amplified over extensive time periods in vitro without diminution of their long-term in vivo repopulating potential. These results also set the stage for analogous studies of human stem cell selection and expansion in vitro, which may be important for future gene therapy protocols.

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PubMed1989

Neonatal W-mutant mice are favorable hosts for tracking development of marked hematopoietic stem cells.

Neonatal unirradiated mice of W-mutant genotypes, with a hematopoietic stem cell defect and anemia, were injected i.v. with normal fetal liver hematopoietic cells. Efficient, long-term engraftment occurred as a result of the competitive advantage to the donor stem cells. The frequency of engraftment and rate of repopulation characteristically diminish in the series W/Wv, Wf/Wf, and Wv/+, in which the severity of the endogenous defect is progressively less. H-2 compatibility is required in the inbred strain combinations examined; other histocompatibility loci play a minor role in some strain combinations. Engraftment is due to self-renewing hematopoietic stem cells ancestral to myeloid and lymphoid lineages. The more mildly defective mutants display much greater variability in the kinetics of repopulation--a result consistent with seeding by single, or very few, stem cells that form developing clones. Engraftment efficiency is reduced by prolonged culture of fetal liver cells during experimental infection by recombinant retroviruses; nevertheless, after 24 h in vitro to achieve retroviral marking, stem cells retain their ability to repopulate and develop in W/Wv neonates.

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PubMed1989

Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells.

The effects of several hematopoietic growth factors on primitive murine bone marrow progenitor cells [colony-forming unit(s)-spleen (CFU-S)] have been investigated during culture for 2-6 days. Interleukin 3 (IL-3) was required for CFU-S survival in culture, and the combination of IL-3 and interleukin 6 (IL-6) increased the number of CFU-S in culture 10-fold over the number obtained with IL-3 alone. Stem cell function was measured by competitive repopulation; IL-3 was required, and IL-3 and IL-6 appear to act synergistically to enhance stem cell recovery from these cultures. These data appear to be relevant for retroviral-mediated gene transfer into stem and progenitor cells. Murine bone marrow cells were infected with a retrovirus containing the human beta-globin gene in the presence of various growth factors. Only 2 of 17 mice reconstituted with cells infected in the presence of IL-3 alone showed long-term expression of the human beta-globin gene (12 months), as opposed to 6 of 11 mice reconstituted with cells infected in the presence of IL-3 and IL-6. Medium conditioned by 5637 bladder carcinoma cells, a source of several hematopoietic growth factors, increased the frequency of infection of CFU-S but did not enhance stem cell infection or the repopulating potential of cultured bone marrow cells. Stem cells containing the human beta-globin provirus from these animals were shown to be capable of reconstituting secondary recipients in which the human beta-globin gene was expressed.

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PubMed1996

Hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo.

The hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood (UCB) samples was evaluated in vitro and in vivo. Phenotypic analysis shows that approximately 1% of cord blood mononuclear cells express high levels of CD34 antigen on their surface (CD34hi), but none of a panel of lineage antigens (Lin-), suggesting that they are hematopoietic progenitor cells that have not yet committed to a specific lineage. Approximately 1% of CD34hi/Lin- cells are primitive hematopoietic progenitors that produce B lymphoid and multiple myeloid progeny for up to 7 weeks in stromal cell cultures. Twenty-one percent (+/- 13%) of CD34hi/Lin- cells also express low levels of the Thy-1 antigen and are threefold to fourfold enriched over CD34hi/Lin- cells in primitive hematopoietic potential as measured by long-term culture and phenotypic analysis. One-week liquid cultures of CD34-enriched UCB progenitor cells in the presence of interleukin (IL)-3, IL-6, and stem cell factor (SCF) results in a two-fold to threefold expansion of progenitors capable of reinitiating long-term stromal cell cultures. Only the CD34hi/Thy-1+/Lin- cell population was capable of maintaining progenitors with secondary transfer potential in long-term stromal cell cultures and is thus postulated to contain all of the primitive hematopoietic stem cells in UCB. The in vivo transplantation potential of UCB was also measured. Ex vivo manipulated UCB progenitor cells were used to engraft irradiated human thymus fragments implanted in severe combined immunodeficiency (SCID) mice. Thymic engraftment with >5% donor-derived cells and a normal CD4/CD8 distribution was observed in 19 of 23 tissues tested. UCB cells from in vitro expansion cultures engrafted with efficiencies comparable to nonexpanded cells. Similar results were obtained for UCB engraftment of human bone fragments implanted in SCID mice. In all cases, engraftment was achieved in competition with endogenous competitor stem cells and across major histocompatibility barriers. Taken together, this data demonstrates that human UCB is a rich source of multipotent hematopoietic progenitors that can be cryopreserved, enriched by physical methods, and expanded in a limited fashion without measurable loss of long-term culture or in vivo engrafting potential as measured in these assays.

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PubMed1996

Proliferation of multipotent hematopoietic cells controlled by a truncated erythropoietin receptor transgene.

The long-term efficacy of gene therapy using bone marrow transplantation requires the engraftment of genetically altered totipotent hematopoietic stem cells (THSCs). Ex vivo expansion of corrected THSCs is one way to increase the efficiency of the procedure. Similarly, selective in vivo expansion of the therapeutic THSCs rather than the endogenous THSCs could favor the transplant. To test whether a conferred proliferative advantage gene can facilitate the in vitro and in vivo expansion of hematopoietic stem cells, we have generated transgenic mice expressing a truncated receptor for the growth factor erythropoietin. These mice are phenotypically normal, but when treated in vivo with exogenous erythropoietin they exhibit a marked increase in multipotent, clonogenic hematopoietic cells [colony-forming units in the spleen (CFU-S) and CFUs that give rise to granulocytes, erythroid cells, macrophages, and megakaryocytes within the same colony (CFU-GEMM)] in comparison with the wild-type mice. In addition, long-term in vitro culture of tEpoR transgenic bone marrow in the presence of erythropoietin induces exponential expansion of trilineage hematopoietic stem cells not seen with wild-type bone marrow. Thus, the truncated erythropoietin receptor gene shows promise as a means for obtaining cytokine-inducible hematopoietic stem cell proliferation to facilitate the direct targeting of THSCs and to provide a competitive repopulation advantage for transplanted therapeutic stem cells.

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PubMed2005

Competitive equality of donor cells expressing a disparate MHC antigen following stem cell-enriched bone marrow transplantation.

INTRODUCTION: Bone marrow cells expressing foreign MHC antigens survive poorly after transplantation. Stable mixed hematopoietic chimerism requires reconstitution with a relatively large number of foreign bone marrow cells and intensive depletion of host cells. In addition, when foreign MHC-transduced autologous bone marrow cells are transplanted, prolonged hematopoietic transgene expression requires extensive host conditioning. The competitive disadvantage associated with engraftment of donor cells expressing foreign MHC antigens is thought to result from a defect in engraftment secondary to donor-host incompatibility or immunologic resistance by the host. METHODS: We used a limiting-dilution competitive repopulation assay with cells from HLA-A2.1 transgenic mice to determine whether and to what extent foreign MHC antigen expression impairs engraftment in C57BL/6 hosts. Transplants were performed with Hoechst 33342 fluorescence-sorted side population (SP) cells, a subset of bone marrow enriched for stem cells. RESULTS.: Transplantation with 250 stem cell-enriched HLA-A2.1-transgenic side population cells successfully competed with nearly 5000 host C57BL/6 side population cells to produce stable long-term mixed chimerism. There was a direct relationship between the number of transplanted donor HLA-A2-expressing cells and the percentage of HLA-A2-expressing cells in the peripheral blood of reconstituted C57BL/6 mice (r2=0.1799, P=0.031). This correlation was maintained in secondary transplant recipients. CONCLUSIONS: HLA-A2-expressing hematopoietic cells do not have an engraftment defect when transplanted into C57BL/6 hosts and immunologic resistance did not limit chimerism following lethal irradiation. These results may have relevance to understanding long-term gene expression after hematopoietic stem cell based gene therapy.

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PubMed1985

Two subpopulations of stem cells for T cell lineage.

An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells, the generation of donor-derived T cells being observed in two out of 14 recipients transferred with as few as 1.5 X 10(4) cells. The stem cell activity of spleen cells was estimated to be about 1% of that of bone marrow cells, and no activity was found in thymus cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. Spleen cells showed a markedly high level of activity 7 days after the reconstitution, followed by a decline, whereas the activity of bone marrow cells was very low on day 7 and increased crosswise. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells. Such patterns of compartmentalization of stem cells in the spleen and bone marrow of irradiated recipients completely conform to the general scheme of the relationship between restricted stem cells and less mature stem cells, including pluripotent stem cells, which became evident in other systems such as in the differentiation of spleen colony-forming cells or of stem cells for B cell lineage.

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PubMed2005

Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells.

Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix.

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