Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150- fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.     

The marrow homing efficiency of murine hematopoietic stem cells remains constant during ontogeny

OBJECTIVE: Several recent studies have established the potential clinical utility of hematopoietic stem cells (HSCs) not only for marrow rescue but also for regenerating diseased or damaged nonhematopoietic tissues. These findings have focused renewed interest in understanding the in vivo trafficking patterns of HSCs from different sources. Previous experiments have suggested that the half-life of HSCs in the circulation is short, although the actual proportion that return to the bone marrow (BM) following transplantation has not been previously quantitated. The present study was undertaken to measure this fraction and compare the values obtained for functionally defined HSCs from adult murine BM and day-14 fetal liver (FL). METHODS: The number of HSCs that could be recovered from the BM of lethally irradiated mice 24 hours after intravenous injection of Ly-5 congenic BM or FL cells was determined by limiting-dilution competitive repopulating unit (CRU) assays in secondary mice. RESULTS: The marrow seeding efficiency of both adult BM- and FL-CRU able to produce lymphoid and myeloid progeny for 5-26 weeks posttransplant was approximately 10%. FL-CRU generated clones that were approximately threefold larger than those produced by BM-CRU. Interestingly, clones produced by "homed" HSCs were approximately twofold smaller than those produced by freshly isolated HSCs. Differences were also seen in the proportions of lymphoid vs myeloid progeny generated by fresh and homed HSCs. CONCLUSIONS: These data suggest common mechanisms regulating the BM homing of long-term repopulating HSCs throughout ontogeny despite subtle differences in the size and composition of the clones they generate.     

Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell- depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.     

Lymphoid reconstitution after transplantation of congenic hematopoietic cells in busulfan-treated mice.

The effects of pretransplant conditioning with high-dose busulfan, a myeloablative but nonimmunosuppressive alkylating agent, on reconstitution of lymphoid tissues by donor cells after bone marrow transplantation (BMT) has not been extensively examined. We used flow cytometric analyses to study the kinetics and extent of lymphocyte repopulation in C57BL/6 mice (immunophenotype Ly-5.2) given graded doses of busulfan (10 to 100 mg/kg) or total body irradiation (TBI; 900 rad) and hematopoietic cell transplantation (HCT; transplantation of bone marrow and spleen cells) from congenic Ly-5.1 donors. Mice transplanted after 10 mg/kg of busulfan had slow and incomplete lymphoid engraftment; only 6% to 11% of lymphocytes in the peripheral blood, lymph nodes, and spleen were positive for Ly-5.1 at 30 days after transplant, slightly increased to 13% to 20% at 60 days, and stabilized at 40% to 46% by 180 days after HCT. Higher doses of busulfan (20 to 100 mg/kg) provided dose-dependent congenic lymphoid reconstitution. Thirty days after HCT, the range of Ly-5.1 cells in blood, lymph nodes, and spleen of Ly-5.2 recipient mice was 43% to 54% after 20 mg/kg of busulfan, 66% to 71% after 50 to 80 mg/kg, and 77% to 85% after 100 mg/kg. Sixty days after transplant, lymphoid chimerism increased to 57% to 68% in 20 mg/kg recipients, 72% to 79% after 35 mg/kg, and 75% to 90% in animals given 50 mg/kg or greater, as seen in radiation chimeras. Despite slower early reconstitution after lower doses of busulfan, donor lymphocytes exceeded 90% to 95% by 90 to 120 days after HCT in all mice given at least 20 mg/kg. Even though busulfan lacks directly immunosuppressive properties, virtually complete sustained lymphoid reconstitution by transplanted congenic donor stem cells occurs after its administration. These observations suggest that pretreatment with busulfan may be effective in gene therapy strategies that involve infusion of autologous marrow cells into which functional genes have been inserted.     

Reversible expression of CD34 by murine hematopoietic stem cells.

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34(-). We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34(-) and CD34(+) cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage(-) c-kit(+) Sca-1(+) CD34(-) bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34(-) and CD34(+) cell populations and tested for their engraftment capabilities, only CD34(+) cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34(+) stem cells to CD34(-) state. We transplanted Ly-5.1 CD34(+) post-5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34(-) cell fraction, indicating the reversion of CD34(+) to CD34(-) stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.     

Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells

Attempts to maintain or expand primitive hematopoietic stem cells in vitro without the concomitant loss of their differentiative and proliferative potential in vivo have largely been unsuccessful. To investigate this problem, we compared the ability of three cloned bone marrow (BM) stromal cell lines to support the growth of primitive Thy- 1lo Sca-1+H-2Khi cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly enriched in cobblestone area-forming cells (CAFC), but their frequency was dependent on the stromal cell lines used in this assay (1 per 45 cells on SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8 cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high clonogenicity of this primitive stem cell population. More primitive stem cells with competitive repopulating potential were measured by injecting the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5) radioprotective Ly-5.1 BM cells whose long-term repopulating ability has been "compromised" by two previous cycles of marrow transplantation and regeneration. Donor-derived lymphocytes and granulocytes were detected in 66% of animals injected with 50 sorted cells. To quantitate the maintenance of competitive repopulating units (CRU) by stromal cells, sorted cells were transplanted at limiting dilution before and after being cultured for 2 weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per 55 freshly sorted cells. CRU could be recovered from cocultures supported by all three stromal cell lines, but their numbers were approximately-sevenfold less than on day 0. In contrast, the addition of LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (approximately two-fold to threefold decline), and enabled their maintenance on SyS-1. LIF appeared to act indirectly, because alone it did not support the proliferation of Thy- 1lo Sca-1+H-2Khi cells in stroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1 within 1 to 6 hours of LIF-stimulation. To determine if increased expression of SLF by LIF-stimulated SyS-1 cells could account for their capacity to support stem cells, sorted calls were cocultured on simian CV-E cells that were transfected with an expression vector encoding membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF synergized with IL-6 produced endogenously by CV-E cells enabling CAFC growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF- stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence for a role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, we have used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture.     

Bone marrow origin of hematopoietic progenitors and stem cells in murine muscle

It has been reported that mononuclear cells harvested from murine skeletal muscle are capable of hematopoietic reconstitution of lethally irradiated mice. First, the nature of the hematopoietic progenitors in the muscle of C57BL/6-Ly-5.1 mice was examined by means of methylcellulose culture. The types and incidences of colonies grown from muscle mononuclear cells were different from those cultured from bone marrow (BM) or peripheral blood mononuclear cells. The next step was to examine the origin of the hematopoietic progenitors and stem cells in the muscle with the use of Ly-5.2 mice that had been made chimeric by transplantation of Ly-5.1 BM cells. The percentages of Ly-5.1 cells cultured from the muscle of the chimeric mice correlated with those cultured from BM, indicating BM origin of hematopoietic progenitors in the muscle. Long-term hematopoietic engrafting cells in the muscle of the chimeric mice were also derived from BM. However, mobilization of progenitors into circulation by granulocyte colony-stimulating factor did not change the population of hematopoietic progenitors in the muscle. It is proposed that hematopoietic progenitors and stem cells in the muscle tissue are of BM origin but their transition from BM to muscle may be a slow process.     

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.     

Development of intraepithelial T lymphocytes in the intestine of irradiated SCID mice by adult liver hematopoietic stem cells from normal mice

BACKGROUND/AIMS: We recently reported the adult mouse liver to contain c-kit+ stem cells that can give rise to multilineage leukocytes. This study was designed to determine whether or not adult mouse liver stem cells can generate intraepithelial T cells in the intestine as well as to examine the possibility that adult liver c-kit+ stem cells originate from the fetal liver. METHODS: Adult liver mononuclear cells, bone marrow (BM) cells, liver c-kit+ cells or bone BM c-kit+ cells of BALB/c mice were i.v. transferred into 4 Gy irradiated CB17/-SCID mice. In other experiments, fetal liver cells from Ly5.1 C57BL/6 mice and T cell depleted adult BM cells from Ly5.2 C57BL/6 mice were simultaneously transferred into irradiated C57BL/6 SCID mice (Ly5.2). At 1 to 8 weeks after cell transfer, the SCID mice were examined. RESULTS: Not only BM cells and BM c-kit+ cells but also liver mononuclear cells and liver c-kit+ cells reconstituted gamma delta T cells, CD4+ CD8+ double-positive T cells and CD8 alpha+beta- T cells of intestinal intraepithelial lymphocytes of SCID mice. Injection of a mixture of fetal liver cells from Ly5.1 C57BL/6 mice and adult BM cells from Ly5.2 C57BL/6 mice into Ly5.2 C57BL/6 SCID mice induced both Ly5.1 and Ly5.2 T cells, while also generating c-kit+ cells of both Ly5.1 and Ly5.2 origins in the liver. CONCLUSIONS: Adult mouse liver stem cells were able to generate intestinal intraepithelial T cells of the SCID mice, and it is thus suggested that some adult liver stem cells may indeed be derived from the fetal liver.     

Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow.

The purpose of this study was to develop a simple assay for quantitating transplantable human lymphomyeloid stem cells (competitive repopulating units [CRU]) to enable comparison among the numbers and types of progeny generated in NOD/ SCID mice by such cells from different ontologic sources. Sub-lethally irradiated NOD/SCID mice were transplanted with varying numbers of CD34+ cell-enriched suspensions of human fetal liver, cord blood, or adult marrow cells. The types and numbers of human cells present in the marrow of the mice were measured 6 to 8 weeks later using flow cytometry, in vitro progenitor assays, and secondary transplant endpoints. Frequencies of human CRU obtained by limiting dilution analysis of mice repopulated 6 to 8 weeks posttransplant were the same when the lymphoid and myeloid progeny of CRU were both detected by specific immunophenotypic endpoints as when in vitro myeloid progenitor assays were used to detect CRU myelopoietic activity. The average output per injected CRU of very primitive cells (CD34(+)CD38(-) cells, LTC-IC, and secondary CRU) was found to be highest for fetal liver CRU and progressively decreased (up to >100-fold) for ontologically older CRU. In contrast, the average output of mature cells was highest for cord blood CRU and lowest for fetal liver CRU, despite equivalent production of intermediate progenitors. Differences in the relative numbers of mature lymphoid, myeloid, and erythroid progeny produced by CRU from different ontologic sources were also seen. Finally, evidence of a transplantable human lymphoid-restricted cell present throughout ontogeny was obtained. A simpler and easier assay for enumerating transplantable human stem cells with lymphomyeloid reconstituting activity has been described, and its specificity and sensitivity validated. The use of this assay has revealed ontogeny-associated differences in a variety of functional attributes of human stem cells proliferating and differentiating in an in vivo, but xenogeneic, setting.     

Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells

To test the hypothesis that aging has negative effects on stem-cell homing and engraftment, young or old C57BL/6 bone marrow (BM) cells were injected, using a limiting-dilution, competitive transplantation method, into old or young Ly5 congenic mice. Numbers of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) recovered from BM or spleen were measured and compared with the numbers initially transplanted. Although the frequency of marrow competitive repopulation units (CRUs) increased approximately 2-fold from 2 months to 2 years of age, the BM homing efficiency of old CRUs was approximately 3-fold lower than that of young CRUs. Surprisingly, the overall size of individual stem-cell clones generated in recipients receiving a single CRU was not affected by donor age. However, the increased ages of HSC donors and HSC transplant recipients caused marked skewing of the pattern of engraftment toward the myeloid lineage, indicating that HSC-intrinsic and HSC-extrinsic (microenvironmental) age-related changes favor myelopoiesis. This correlated with changes after transplantation in the rate of recovery of circulating leukocytes, erythrocytes, and platelets. Recovery of the latter was especially blunted in aged recipients. Collectively, these findings may have implications for clinical HSC transplantation in which older persons increasingly serve as donors for elderly patients.     

Regulatory elements of the vav gene drive transgene expression in hematopoietic stem cells from adult mice

OBJECTIVE: Previous studies have shown that the HS21/45 promoter of the vav protooncogene drives a predominant expression of exogenous transgenes in mouse hematopoietic cells, including clonogenic bone marrow (BM) progenitors. We investigated the activity of this promoter in the hematopoietic stem cell compartment of adult mice. MATERIALS AND METHODS: Inbred Ly5.1 transgenic mice expressing a nonfunctional human CD4 marker gene (hCD4) under the control of the HS21/45 promoter were generated. BM cells from these animals were sorted based on the intensity of hCD4 expression. Fractions characterized by high, intermediate, or low/negative expression of the transgene were then assessed for their competitive repopulation ability (CRA), using unfractionated BM cells from Ly5.2 mice as a reference competitor population. RESULTS: Data showed that BM cells having a low/negative or intermediate expression of hCD4 had a very poor hematopoietic CRA. In contrast, BM cells with high hCD4 expression were characterized by a high CRA. These observations were confirmed in the short- and long-term posttransplantation of primary and secondary recipients when analyzing the lymphoid and myeloid cells of recipient mice. CONCLUSIONS: Our results demonstrate for the first time that the regulatory HS21/45 sequence of the vav gene constitutes an efficient promoter for driving transgene expression in multipotent hematopoietic stem cells residing in the BM of adult mice. Thus, this promoter is proposed for the development of transgenic mice and gene therapy vectors that require restricted expression of exogenous transgenes in cells of the hematopoietic system, including primitive hematopoietic stem cells.     

CD34 expression by murine hematopoietic stem cells mobilized by granulocyte colony-stimulating factor

Controversy has existed about CD34 expression by hematopoietic stem cells. We recently reported that CD34 expression reflects the activation state of stem cells by using a murine transplantation model. It has been generally held that mobilized blood stem cells express CD34.However, it has also been reported that mobilized stem cells and progenitors are in G0/G1 phases of the cell cycle. To address the state of CD34 expression by the mobilized stem cells, we again used the mouse transplantation model. We prepared CD34(-) and CD34(+) populations of nucleated blood cells from granulocyte colony-stimulating factor-treated Ly-5.1 mice and assayed each population for long-term engrafting cells in lethally irradiated Ly-5.2 mice. The majority of the stem cells were in the CD34(+) population. The CD34 expression by mobilized stem cells was reversible because re-transplantation of Ly-5.1 CD34(-) marrow cells harvested from the Ly-5.2 recipients of CD34(+)-mobilized stem cells 8 months posttransplantation revealed long-term engraftment. These results may support the use of total CD34(+) cells in mobilized blood as a predictor for engraftment and CD34 selection for enrichment of human stem cells. (Blood. 2000;96:1989-1993)     

Distinct changes in adult lymphopoiesis in Rag2-/- mice fully reconstituted by alpha4-deficient adult bone marrow cells

OBJECTIVE: alpha4 Integrins are major players in lymphoid cell trafficking and immune responses. However, their importance in lymphoid reconstitution and function, studied by antibody blockade or in genetic models of chimeric animals with alpha4(KO) embryonic stem (ES) cells, competitive repopulation experiments with fetal liver(KO) cells, or in beta1/beta7 doubly-deficient mice has yielded disparate conclusions. MATERIALS AND METHODS: To study the role of alpha4 integrin (alpha4beta1, alpha4beta7) during adult life, we transplanted lethally irradiated Rag2(-/-) mice with alpha4(Delta/Delta) or alpha4(f/f) adult bone marrow (BM) cells and evaluated recipients at several points after transplantation. RESULTS: Lymphomyeloid repopulation (8 months later) was entirely donor-derived in all recipients, and novel insights regarding lymphoid reconstitution and function were revealed. Thymic repopulation was impaired in all alpha4(Delta/Delta) recipients, likely because of homing defects of BM-derived progenitors, although a role of alpha4 integrin in intrathymic expansion/maturation of T cells cannot be excluded; reconstitution of gut lymphoid tissue was also greatly diminished because of homing defects of alpha4(Delta/Delta) cells; impaired immunoglobulin (Ig) M and IgE, but normal IgG responses were seen, suggesting compromised initial B-/T-cell interactions, whereas interferon-gamma production from ovalbumin-stimulated cells was increased, possibly reflecting a bias against Th2 stimulation. CONCLUSION: These data complement previous observations by defending the role of alpha4 integrin in thymic and gut lymphoid tissue homing, and by strengthening evidence of attenuated B-cell responses in alpha4-deficient mice.     

Expansion of hematopoietic stem cells in the developing liver of a mouse embryo

The activity of hematopoietic stem cells in the developing liver of a C57BL/6 mouse embryo was quantified by a competitive repopulation assay. Different doses of fetal liver cells at days 11 to 18 of gestation were transplanted into irradiated mice together with 2 x 10(5) adult bone marrow cells. A long-term repopulation in myeloid-, B-cell, and T-cell lineage by fetal liver cells was evaluated at 20 weeks after transplantation. At day 12 of gestation multilineage repopulating activity was first detected in the liver as 50 repopulating units (RU) per liver. The number of RU per liver increased 10-fold and 33-fold by day 14 and day 16 of gestation, and decreased thereafter, suggesting a single wave of stem cell development in the fetal liver. A limiting dilution analysis revealed that the frequency of competitive repopulating units (CRU) in fetal liver cells at day 12 of gestation was similar to that at day 16 of gestation. Because of an increase of total fetal liver cell number, the absolute number of CRU per liver from days 12 to 16 of gestation increased 38-fold. Hence, the mean activity of stem cells (MAS) that is given by RU per CRU remained constant from days 12 to 16 of gestation. From these data we conclude that hematopoietic stem cells expand in the fetal liver maintaining their level of repopulating potential.     

In utero transplantation of human fetal haemopoietic cells in NOD/SCID mice

We have previously demonstrated that high levels of allogeneic, donor-derived mouse haemopoietic progenitor cells engraft following in utero transplantation in NOD/SCID mice. To evaluate whether the fetal NOD/SCID haemopoietic microenvironment supports the growth and development of human fetal haemopoietic progenitor cells, we injected fetal liver mononuclear cells (FL) or fetal bone marrow (FBM) derived CD34⁺ cells into NOD/SCID mice on day 13/14 of gestation. At 8 weeks of age 12% of FBM recipients and 10% of FL recipients were found to have been successfully engrafted with CD45⁺ human cells. CD45⁺ cells were present in the BM of all chimaeric animals; 5/6 recipients showed engraftment of the spleen, and 4/6 recipients had circulating human cells in the peripheral blood (PB). The highest levels of donor cells were found in the BM, with up to 15% of the nucleated cells expressing human specific antigens. Multilineage human haemopoietic engraftment, including B cells (CD19), myelomonocytic cells (CD13/33) and haemopoietic progenitor cells (CD34), was detected in the BM of chimaeric mice. In contrast, no human CD3⁺ cells were detected in any of the tissues evaluated. When the absolute number of engrafted human cells in the PB, BM and spleens of chimaeric mice was determined, a mean 16-fold expansion of human donor cells was observed. Although multilineage engraftment occurs in these fetal recipients, both the frequency and the levels of engraftment are lower than those previously reported when human cells are transplanted into adult NOD/SCID recipients.     

Reactivity of murine cytokine fusion toxin, diphtheria toxin390-murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor cells

Myeloid leukemias can express interleukin-3 receptors (IL-3R). Therefore, as an antileukemia drug, a fusion immunotoxin was synthesized consisting of the murine IL-3 (mIL-3) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mIL-3 hybrid gene was cloned into a vector under the control of an inducible promote. The fusion protein was expressed in Escherichia coli and then purified from inclusion bodies. The fusion toxin was potent because it inhibited FDC-P1, an IL-3R- expressing murine myelomonocytic tumor line (IC50 = 0.025 nmol/L or 1.5 ng/mL). Kinetics were rapid and cell-free studies showed that DT390-mIL- 3 was as toxic as native DT. DT390-mIL-3 was selective because anti-mIL- 3 monoclonal antibody, but not irrelevant antibody, inhibited its ability to kill. Cell lines not expressing IL-3R were not inhibited by the fusion protein. Because the use of DT390-mIL-3 as an antileukemia agent could be restricted by its reactivity with committed and/or primitive progenitor cells, bone marrow (BM) progenitor assays were performed. DT390-mIL-3 selectively inhibited committed BM progenitor cells as measured by in vitro colony-forming unit-granulocyte- macrophage and in vivo colony-forming unit-spleen colony assays. To determine if this fusion protein was reactive against BM progenitor cells required to rescue lethally irradiated recipients, adoptive transfer experiments were performed. Eight million DT390-mIL-3-treated C57BL/6 Ly5.2 BM cells, but not 4 million, were able to rescue lethally irradiated congenic C57BL/6 Ly5.1 recipients, suggesting that progenitor cells might be heterogenous in their expression of IL-3R. This idea was supported in competitive repopulation experiments in which DT390-mIL-3 treated C57BL/6 Ly5.2 BM cells were mixed with nontreated C57BL/6 Ly5.1 BM cells and used to reconstitute C57BL/6 Ly5.1 mice. A significant reduction, but not elimination, of Ly5.2- expressing cells 95 days post-BM transplantation and secondary transfer experiments indicated that IL-3R is not uniformly expressed on all primitive progenitor cells. The fact that some early progenitor cells survived DT390-mIL-3 treatment indicates that this fusion toxin may be useful in the treatment of myeloid leukemias that express the IL-3R.     

Lymphoid and erythroid repopulation in B6 W-anemic mice: a new unirradiated recipient

The W-anemic family of mouse mutants is an important model for studying repopulation in unirradiated recipients. This is the first study of blood lymphoid cell repopulation in adult W-anemic mutants given high doses of marrow cells, and it shows a wide difference in repopulation rates of circulating lymphoid and erythroid cells. This study also offers an improved model for marrow transplantation, using W alleles that are spontaneous mutations on the widely used inbred strain C57BL/6J (B6). Unirradiated B6-W41J/W41J or -W41J/W39J recipients of 2 x 10(6) B6 marrow cells are completely repopulated with donor erythrocytes after 3 months, whereas complete repopulation of lymphocytes requires a year. Surprisingly, the eventual degree of repopulation is independent of the severity of the mutation. The new mutants are not as anemic as the commonly used WBB6F1-W/Wv anemic mutants, they have a much higher ability to form macroscopic spleen colonies (spleen colony-forming units, CFU-S), and B6-W41J/W41J mice are fertile. Nevertheless, lymphoid and erythroid repopulation occur to a similar extent in B6-W41J/W41J or -W41J/W39J and in WBB6F1-W/Wv anemic mutants. Repopulation is more rapid in the latter, but host cells may be damaged by B6 reactions against the WB parent. Avoiding graft-versus-host reactions, hybrid resistance, and similar complications are important advantages in using donors and unirradiated recipients all on the B6 mouse genetic background. Additionally, congenic B6 mice provide a variety of genetic markers, allowing myeloid and lymphoid repopulation to be readily quantitated.     

Enrichment and functional characterization of Sca-1+WGA+, Lin-WGA+, Lin- Sca-1+, and Lin-Sca-1+WGA+ bone marrow cells from mice with an Ly-6a haplotype

Approximately 4% to 5% of all bone marrow (BM) cells and 8% to 9% of low density BM cells from FVB/N and BALB/c mice (Ly-6a haplotype) show high to intermediate expression of Ly-6E.1 antigen, recognized by the Sca-1 antibody. Functional properties of enriched cells expressing Ly- 6E.1-allelic form of Sca-1 antigen were analyzed and correlated with the properties of cells expressing the carbohydrate binding sites for the lectin wheat-germ agglutinin (WGA). Using equilibrium density centrifugation and fluorescence-activated cell sorting, Sca-1+WGA+, Lin- WGA+, Lin-Sca-1+, and Lin-Sca-1+WGA+ cells were isolated and their splenic colony-forming unit (CFU-S) cell content, radioprotection ability, and long-term reconstitution capacity determined. Enriched Sca- 1+WGA+, Lin-WGA+, Lin-Sca-1+ and Lin-Sca-1+WGA+ cells gave rise to 1 CFU-S12 cell out of 26, 20, 21, and 15 sorted cells, respectively. When transplanted into lethally irradiated recipients (100 to 500 cells/mouse) all populations rescued 70% to 100% of recipients in a 30- day radioprotection assay and mediated survival of 40% to 80% of recipients 6 months after transplantation. Using transgenic mice as cell donors we have shown that 12 to 16 weeks after transplantation of 100 Sca-1+WGA+, Lin-WGA+, Lin-Sca-1+, and Lin-Sca-1+WGA+ cells, 40% to 80% of recipients had donor cells in BM, spleen, thymus, and lymph nodes. These results indicate that the population of cells expressing Ly-6E.1 form of Sca-1 antigen in two analyzed mouse strains with Ly-6a haplotype contains CFU-S and long-term repopulating cells. Furthermore, the data suggest that, at least in FVB/N mice, day-12 CFU-S cells and cells with long-term repopulating capacity simultaneously express Ly- 6E.1 form of Sca-1 antigen and WGA-binding molecules.