Highly efficient ex vivo expansion of human hematopoietic stem cells using Delta1-Fc chimeric protein

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy, and clinical transplantation. Here, we demonstrate efficient ex vivo expansion of HSCs measured by long-term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133-sorted cells using a soluble form of Delta1. After a 3-week culture on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, Flt-3 ligand, interleukin (IL)-3, and IL-6/soluble IL-6 receptor chimeric protein (FP6) in a serum- and stromal cell-free condition, we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self-renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/gammac(null) mice, which showed myeloid, B, T, and natural killer cells as well as CD133(+)CD34(+) cells, and hematopoietic reconstitution in the secondary recipients. Interestingly, the CD133-sorted cells contained approximately 4.5 times more SRCs than the CD34-sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.     

Comparison of hematopoietic activities of human bone marrow and umbilical cord blood CD34 positive and negative cells.

Although the hematopoietic activities of human CD34+ bone marrow (BM) and cord blood (CB) cells have been well characterized, the phenotype of nonobese-diabetic severe combined immunodeficient (NOD/SCID) mice repopulating cells (SRCs) in CB and BM has not yet been fully examined. To address this issue, various hematopoietic activities were compared in terms of total and CD34+ CB and BM cells. Clonal culture of fluorescence-activated cell sorter (FACS) CD34+ CB and BM cells revealed a higher incidence of colony-forming cells with greater proliferation capacity in CB over BM CD34+ cells. CB CD34+ cells also demonstrated higher secondary plating efficiency over BM cells. In addition, we demonstrated that mice transplanted with CB mononuclear cells (MNCs) showed significantly higher levels of chimerism than those transplanted with BM MNCs. However, recipients of FACS-sorted CD34+ CB cells showed significantly lower levels of chimerism than those that received total CB MNCs, suggesting a role of facilitating cells in the CD34- cell population. To further analyze the role of CD34- cells, the NOD/SCID repopulating ability of FACS-sorted CB CD34-c-kit+Lin- and CD34-c-kit-Lin- cells were examined. However, SRCs were not detected in those cells. Taken together, these data suggest that CB is a better source of hematopoietic stem cells and that there are cells in the CD34- fraction that facilitate repopulation of hematopoiesis in the NOD/SCID environment.     

Identification of long-term repopulating potential of human cord blood-derived CD34-flt3- severe combined immunodeficiency-repopulating cells by intra-bone marrow injection

Recently, we have identified human cord blood (CB)-derived CD34-negative (CD34(-)) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003;101:2924). In contrast to murine CD34(-) Kit(+)Sca-1(+)Lineage(-) (KSL) cells, human CB-derived Lin(-)CD34(-) cells did not express detectable levels of c-kit by flow cytometry. In this study, we have investigated the function of flt3 in our identified human CB-derived CD34(-) SRCs. Both CD34(+)flt3(+/-) cells showed SRC activity. In the CD34(-) cell fraction, only CD34(-)flt3(-) cells showed distinct SRC activity by IBMI. Although CD34(+)flt3(+) cells showed a rather weak secondary repopulating activity, CD34(+)flt3(-) cells repopulated many more secondary recipient mice. However, CD34(-)flt3(-) cells repopulated all of the secondary recipients, and the repopulating rate was much higher. Next, we cocultured CD34(-)flt3(-) cells with the murine stromal cell line HESS-5. After 1 week, significant numbers of CD34(+)flt3(+/-) cells were generated, and they showed distinct SRC activity. These results indicated that CB-derived CD34(-)flt3(-) cells produced CD34(+)flt3(-) as well as CD34(+)flt3(+) SRCs in vitro. The present study has demonstrated for the first time that CB-derived CD34(-) SRCs, like murine CD34(-) KSL cells, do not express flt3. On the basis of these data, we propose that the immunophenotype of very primitive long-term repopulating human hematopoietic stem cells is Lin(-)CD34(-)c-kit(-)flt3(-). Disclosure of potential conflicts of interest is found at the end of this article.     

Mesenchymal stem cells secreting angiopoietin-like-5 support efficient expansion of human hematopoietic stem cells without compromising their repopulating potential

Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34(+) CD133(+) cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded ~60-fold by limiting dilution assay in NOD-scid Il2rg(-/-) (NSG) mice. The cultured CD34(+) CD133(+) cells had similar engraftment potential to uncultured CD34(+) CD133(+) cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34(+) CD133(+) cells maintain both short-term and long-term HSC activities. To our knowledge, this ~60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.     

Cycling G1 CD34+/CD38+ cells potentiate the motility and engraftment of quiescent G0 CD34+/CD38-/low severe combined immunodeficiency repopulating cells

The mechanism of human stem cell expansion ex vivo is not fully understood. Furthermore, little is known about the mechanisms of human stem cell homing/repopulation and the role that differentiating progenitor cells may play in these processes. We report that 2- to 3-day in vitro cytokine stimulation of human cord blood CD34(+)-enriched cells induces the production of short-term repopulating, cycling G1 CD34(+)/CD38(+) cells with increased matrix metalloproteinase (MMP)-9 secretion as well as increased migration capacity to the chemokine stromal cell-derived factor-1 (SDF-1) and homing to the bone marrow of irradiated nonobese diabetic severe/combined immunodeficiency (NOD/SCID) mice. These cycling G1 cells enhance SDF-1-mediated in vitro migration and in vivo homing of quiescent G0 CD34(+) cells, which is partially abrogated after inhibition of MMP-2/-9 activity. Moreover, the engraftment potential of quiescent G0 SCID repopulating cells (SRCs) is also increased by the cycling G1 CD34(+)/CD38(+) cells. This effect is significantly abrogated after incubation of cycling G1 cells with a neutralizing anti-CXCR4 antibody. Our data suggest synergistic interactions between accessory cycling G1 CD34(+)/CD38(+) committed progenitor cells and quiescent, primitive G0 CD34(+)/CD38(-/low) SRC/stem cells, the former increasing the motility and engraftment potential of the latter, partly via secretion of MMP-9.     

Human progenitor cells rapidly mobilized by AMD3100 repopulate NOD/SCID mice with increased frequency in comparison to cells from the same donor mobilized by granulocyte colony stimulating factor.

AMD3100 inhibits the interaction between SDF-1 and CXCR4, and rapidly mobilizes hematopoietic progenitors for clinical transplantation. However, the repopulating function of human cells mobilized with AMD3100 has not been characterized in comparison to cells mobilized with granulocyte-colony stimulating factor (G-CSF) in the same donor. Therefore, healthy donors were leukapheresed 4 hours after injection with AMD3100; after 10 days of drug clearance the same donor was mobilized with G-CSF, allowing a paired comparison of repopulation by mobilized cells. Transplantation of mononuclear cells (MNC) or purified CD34(+) cells was compared at limiting dilution into NOD/SCID mice. Human AMD3100-mobilized MNC possessed enhanced repopulating frequency in comparison to G-CSF-mobilized MNC from paired donors, and purified CD34(+) progenitors were at least as efficient as the G-CSF mobilized cells. The frequencies of NOD/SCID repopulating cells (SRC) were 1 SRC in 8.7 x 10(6) AMD3100-mobilized MNC compared to 1 SRC in 29.0 x 10(6) G-CSF-mobilized MNC, and 1 SRC in 1.2 x 10(5) AMD3100-mobilized CD34(+) cells compared to 1 SRC in 1.8 x 10(5) G-CSF-mobilized CD34(+) cells. Hematopoietic differentiation of transplanted progenitors was similar after AMD3100 or G-CSF-mobilization. Thus, AMD3100 mobilized peripheral blood represents a rapidly obtained, highly repopulating source of hematopoietic progenitors for clinical transplantation.     

Phenotypic and functional reversal within the early human hematopoietic compartment

The fate of phenotypically defined human hematopoietic stem cells (hHSCs) in culture and the link between their surface marker expression profile and function are still controversial. We studied these aspects of hHSC biology by relating the expression of the early lineage markers (ELM) CD33, CD38, and CD71 on the surface of human umbilical cord blood (UCB) CD34(+) cells to their long-term nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse repopulation activity (LT-SRA). In uncultured UCB samples, LT-SRA was largely confined to the small CD34(+)ELM(-) cell fraction. CD34(+) cells expressing ELM markers at their surface usually lacked LT-SRA. After culturing UCB CD34(+) cells for 6 days in serum-free medium and on a feeder layer of Rat2 cells, the number of CD34(+)ELM(-) cells stayed roughly the same or showed a slight increase and the LT-SRA was preserved, suggesting a close association between LT-SRA and the CD34(+)ELM(-) phenotype. Indeed, transplantation of CD34(+)ELM(-) cells isolated from cultured UCB CD34(+) cells resulted in long-term hematopoietic reconstitution of conditioned NOD/SCID mice, whereas CD34(+)ELM(+) cells derived from the same cultures were devoid of LT-SRA. Remarkably, roughly 1% of the cells recovered from cultures initiated with isolated CD34(+)ELM(+) cells had lost ELM surface expression. Concurrently, the cultured CD34(+)ELM(+) cells acquired LT-SRA, suggesting that hematopoietic stem cells (HSCs) may arise by the dedifferentiation of early hematopoietic progenitor cells. The latter finding challenges the paradigm of unidirectional hematopoietic differentiation and opens new opportunities for HSC expansion prior to transplantation.     

Sustained long-term engraftment and transgene expression of peripheral blood CD34+ cells transduced with third-generation lentiviral vectors

As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34(+) cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34(+) (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB. Disclosure of potential conflicts of interest is found at the end of this article.     

Diprotin A infusion into nonobese diabetic/severe combined immunodeficiency mice markedly enhances engraftment of human mobilized CD34+ peripheral blood cells

Hematopoietic stem cell (HSC) graft cell dose impacts significantly on allogeneic transplant. Similarly, HSC gene therapy outcome is affected by loss of repopulating cells during culture required for ex vivo retrovirus transduction. Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a central role in marrow trafficking of HSCs, and maneuvers that enhance CXCR4 activation might positively impact outcome in settings of limiting graft dose. CD26/dipeptidyl peptidase IV (DPP-IV) is an ectoenzyme protease that cleaves SDF-1, thus reducing CXCR4 activation. We show that injection of irradiated nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with >or=2 micromol Diprotin A (a tripeptide specific inhibitor of CD26 protease activity) at the time of transplant of human granulocyte colony-stimulating factor (G-CSF) mobilized CD34(+) peripheral blood cells (CD34(+) PBCs) results in a >3.4-fold enhancement of engraftment of human cells. We also show that CD26 on residual stromal cells in the irradiated recipient marrow milieu, and not any CD26 activity in the human CD34(+) PBC graft itself, plays the critical role in regulating receptivity of this environment for the incoming graft. Human marrow stromal cells also express CD26, raising the possibility that Diprotin A treatment could significantly enhance engraftment of HSCs in humans in settings of limiting graft dose just as we observed in the NOD/SCID mouse human xenograft model.     

Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells.

So far, blood progenitor cells (BPC) expanded ex vivo in the absence of stromal cells have not been demonstrated to reconstitute hematopoiesis in myeloablated patients. To characterize the fate of early hematopoietic progenitor cells during ex vivo expansion in suspension culture, human CD34(+)-enriched BPC were cultured in serum-free medium in the presence of FLT3 ligand (FL), stem cell factor (SCF) and interleukin 3 (IL-3). Both CD34 surface expression levels and the percentage of CD34+ cells were continuously downregulated during the culture period. We observed an expansion of colony-forming units granulocyte-macrophage (CFU-GM) and BFU-E beginning on day 3 of culture, reaching an approximate 2-log increase by days 5 to 7. Limiting dilution analysis of primitive in vitro clonogenic progenitors was performed through a week 6 cobblestone-area-forming cell (CAFC) assay, which has previously been shown to detect long-term bone marrow culture-initiating cells (LTC-IC). A maintenance or a slight (threefold) increase of week 6 CAFC/LTC-IC was found after one week of culture. To analyze the presence of BPC mediating in vivo engraftment, expanded CD34+ cells were transplanted into preirradiated NOD/SCID mice at various time points. Only CD34+ cells cultured for up to four days successfully engrafted murine bone marrow with human cells expressing myeloid or lymphoid progenitor phenotypes. In contrast, five- and seven-day expanded human BPC did not detectably engraft NOD/SCID mice. When FL, SCF and IL-3-supplemented cultures were performed for seven days on fibronectin-coated plastic, or when IL-3 was replaced by thrombopoietin, colony forming cells and LTC-IC reached levels similar to those of control cultures, yet no human cell engraftment was recorded in the mice. Also, culture in U-bottom microplates resulting in locally increased CD34+ cell density had no positive effect on engraftment. These results indicate that during ex vivo expansion of human CD34+ cells, CFC and LTC-IC numbers do not correlate with the potential to repopulate NOD/SCID mice. Our results suggest that ex vivo expanded BPC should be cultured for limited time periods only, in order to preserve bone-marrow-repopulating hematopoietic stem cells.     

CD26 inhibition on CD34+ or lineage- human umbilical cord blood donor hematopoietic stem cells/hematopoietic progenitor cells improves long-term engraftment into NOD/SCID/Beta2null immunodeficient mice

Given the tremendous need for and potential of umbilical cord blood (CB) to be utilized as a donor source for hematopoietic stem cell (HSC) transplantation in adults, there is a strong push to overcome the constraints created by the limited volumes and subsequent limited HSC and hematopoietic progenitor cell (HPC) numbers available for HSC transplantation from a single collection. We have previously described the use of CD26 inhibitor treatment of donor cells as a method to increase the transplant efficiency of mouse HSCs and HPCs into a mouse recipient. To study the use of CD26 inhibitors as a method of improving the transplantation of human CB HSCs and HPCs, we utilized the nonobese diabetic/severe combined immunodeficient/beta 2 microglobulin null (NOD/SCID/B2m(null)) immunodeficient mouse model of HSC transplantation. We report here significant improvements in the engraftment of long-term repopulating cells following the treatment of either CD34(+) or lineage negative (lin()) donor CB with the CD26 inhibitor, Diprotin A, prior to transplant. These results establish a basis on which to propose the use of CD26 inhibitor treatment of donor CB units prior to transplantation for the purpose of improving transplant efficiency and subsequently patient outcome.     

Quantitative analysis demonstrates expansion of SCID-repopulating cells and increased engraftment capacity in human cord blood following ex vivo culture with human brain endothelial cells

Initial clinical trials examining the transplantation of ex vivo expanded cord blood (CB) cells have failed to demonstrate an impact on hematopoietic recovery compared with historical unmanipulated CB controls. In this study, we tested whether coculture with primary human brain endothelial cells (HUBECs) could increase the engraftment capacity and repopulating cell frequency within CB CD34+ cells. Quantitative analysis demonstrated that HUBEC coculture for 7 days supported a 19-fold greater number of CD34+ cells and 3.4-fold and 2.6-fold greater severe combined immunodeficient (SCID)-repopulating cell (SRC) frequencies than fresh CB CD34+ cells and liquid suspension-cultured cells. Mice transplanted with day-14 HUBEC-cultured cells showed 4.2-fold higher levels of human engraftment than mice transplanted with day-7 HUBEC-cultured cells, indicating that SRC enrichment continued to occur through day 14. Noncontact HUBEC cultures also maintained SRCs at levels comparable with contact HUBEC cultures, demonstrating that HUBEC-secreted soluble factors critically supported SRC self-renewal. Seeding efficiency studies demonstrated that HUBEC-cultured CB CD34+ cells engrafted nonobese diabetic/SCID marrow at significantly higher levels than either fresh CB CD34+ cells or liquid suspension-cultured CD34+ cells. These studies indicate that the application of HUBEC coculture or HUBEC-conditioned media can potentially improve upon current strategies for the clinical expansion of CB stem cells.     

Hematopoietic repopulating ability of cord blood CD34(+) cells in NOD/Shi-scid mice

Although umbilical cord blood (CB) is increasingly being used as an alternative to bone marrow (BM) as a source of transplantable hematopoietic stem cells (HSC), information on the hematopoietic repopulating ability of CB HSC is still limited. We recently established a xenotransplantation system in NOD/Shi-scid mice to evaluate human stem cell activity. In the present study, we transplanted 5 to 10 x 10(4) CB CD34(+) cells into six NOD/Shi-scid mice treated with anti-asialo GM1 antiserum to investigate the hematopoietic repopulating ability of CB. The BM of all recipients contained human CD45(+) cells 10 to 12 weeks after the transplantation (43.8 +/- 17.7%). Clonal culture of the recipient BM cells revealed the formation of various types of human hematopoietic colonies, including myelocytic, erythroid, megakaryocytic, and multilineage colonies, indicating that CB HSC can differentiate into hematopoietic progenitors of various lineages. However, the extent of the differentiation and maturation differed with each lineage. CD13(+)/CD14(+)/CD33(+) myelocytic cells were mainly repopulated in BM and peripheral blood (PB). While CD41(+) megakaryocytic cells and platelets were present, few glycophorin A(+)CD71(+) or hemoglobin alpha-containing erythroid cells were detected. CD19(+) B cells were the most abundantly repopulated in NOD/Shi-scid mice, but their maturational stage differed among the hematopoietic organs. Most of the BM CD19(+) cells were immature B cells expressing CD10 but not surface immunoglobulin (Ig) M, whereas more mature CD19(+)CD10(-) surface IgM(+) B cells were predominantly present in spleen and PB. CD3(+) T cells were not detected even in the recipient thymus. The transplantation to the NOD/Shi-scid mouse may provide a useful tool for evaluating the repopulating ability of transplantable human HSC.     

Inhibition of CD26 in human cord blood CD34+ cells enhances their engraftment of nonobese diabetic/severe combined immunodeficiency mice

CD26, a surface serine dipeptidylpeptidase IV (DPPIV) expressed on different cell types, cleaves the amino-terminal dipeptide from some chemokines, including stromal-derived factor-1 (SDF-1/CXCL12). SDF-1/CXCL12 plays important roles in hematopoietic stem cell (HSC) homing, engraftment, and mobilization. Inhibition of CD26 peptidase activity enhances homing, engraftment, and competitive repopulation in congenic mouse bone marrow cell transplants. Our studies evaluated a role for CD26 in in vivo engraftment of HSCs from human umbilical cord blood (CB) into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Pretreating purified CD34(+) human CB cells with Diprotin A, a DPPIV inhibitor, for 15 min significantly enhanced engraftment. Treatment did not affect differentiation of CD34(+) cells in vivo, as measured phenotypically by human markers CD33, CD38, CD19, and CD34. We found that the percentage of CD26(+) cells within the more immature cells (CD34(+)CD38()) was significantly higher than in the more mature population (CD34(+)CD38(+)). These results suggest that inhibition of CD26 may be one way to enhance engraftment of limiting numbers of stem cells during CB transplantation.     

培养前后脐血CD34~＋细胞在NOD/SCID鼠体内造血重建功能的比较

目的:以NOD/SCID小鼠为模型, 经半致死剂量照射后输注新鲜或培养后的造血细胞, 以比较培养前后脐血CD34 细胞的造血重建功能.方法:从新鲜脐血中分离单个核细胞(MNC), 采用干细胞因子(SCF)、血小板生成素(TPO)、Flt3配体(FL)、白细胞介素3(IL-3)和白细胞介素6(IL-6)细胞因子组合体外培养14 d.通过MiniMACS免疫磁性吸附柱从新鲜或培养后的MNC中分离CD34 细胞, 4×105个CD34 细胞和5×106CD34-细胞混合后通过尾静脉输注入NOD/SCID小鼠中.饲养过程中动态观察外周血象恢复情况, 6周后检测小鼠骨髓和脾脏细胞中人源细胞及各系造血细胞的含量.结果:体外培养MNC 14 d后, 总细胞扩增了1.78倍;细胞移植6周后, 输注新鲜和培养后造血细胞的小鼠均存活, 在小鼠骨髓和脾脏中均可检测到人源细胞及各系人源血细胞和人特异ALU基因序列, 小鼠外周血象恢复到辐照前水平.培养后CD34 细胞在小鼠体内的植入水平与新鲜CD34 细胞的相近, 而其各系人源血细胞的含量高于新鲜CD34 细胞. 结论:体外培养14 d后的CD34 细胞仍保持了体内植入和重建造血的能力, 且其多系造血重建能力优于新鲜CD34 细胞.     

Quiescent human hematopoietic stem cells in the bone marrow niches organize the hierarchical structure of hematopoiesis

Hematopoiesis is a dynamic and strictly regulated process orchestrated by self-renewing hematopoietic stem cells (HSCs) and the supporting microenvironment. However, the exact mechanisms by which individual human HSCs sustain hematopoietic homeostasis remain to be clarified. To understand how the long-term repopulating cell (LTRC) activity of individual human HSCs and the hematopoietic hierarchy are maintained in the bone marrow (BM) microenvironment, we traced the repopulating dynamics of individual human HSC clones using viral integration site analysis. Our study presents several lines of evidence regarding the in vivo dynamics of human hematopoiesis. First, human LTRCs existed in a rare population of CD34(+)CD38(-) cells that localized to the stem cell niches and maintained their stem cell activities while being in a quiescent state. Second, clonally distinct LTRCs controlled hematopoietic homeostasis and created a stem cell pool hierarchy by asymmetric self-renewal division that produced lineage-restricted short-term repopulating cells and long-lasting LTRCs. Third, we demonstrated that quiescent LTRC clones expanded remarkably to reconstitute the hematopoiesis of the secondary recipient. Finally, we further demonstrated that human mesenchymal stem cells differentiated into key components of the niche and maintained LTRC activity by closely interacting with quiescent human LTRCs, resulting in more LTRCs. Taken together, this study provides a novel insight into repopulation dynamics, turnover, hierarchical structure, and the cell cycle status of human HSCs in the recipient BM microenvironment.     

Perforin-independent rejection of transplanted human stem cells

The NOD/SCID mouse model is one of the most established model systems for the analysis of human stem cells in vivo. The lack of mature B and T cells renders NOD/SCID mice susceptible to transplantable human stem and progenitor cells. One remaining functional component of the immune system in NOD/SCID mice is natural killer (NK) cells. We rationalized that by eliminating NK cell-mediated cytotoxicity in this model system engraftment of human haematopoietic stem cells could be improved. Thus perforin-deficient NOD/SCID mice (PNOD/SCID) were generated, which display a complete lack of NK cell-mediated cytotoxicity. To test the engraftment potential of human stem cells in PNOD/SCID mice, we compared the repopulating potential of human haematopoietic stem cells in these mice with the repopulating potential in NOD/SCID mice. Upon injection with varying numbers of mononuclear cells from human cord blood, the number of engrafted PNOD/SCID mice was lower (34.8%) than the number of engrafted NOD/SCID mice (64.7%). Similarly, injection of purified CD34(+) human cord blood cells led to engraftment in 32.3% PNOD/SCID versus 60% in NOD/SCID mice. Surprisingly, these results show that the inactivation of cytotoxic activity of NK cells in PNOD/SCID mice did not result in better engraftment with human haematopoietic stem cells. A potential reason for this observation could be that compensatory activation of NK cells in PNOD/SCID mice induces high levels of soluble factors resulting in an environment unfavourable for human stem cell engraftment.     

The in vivo development of human T cells from CD34(+) cells in the murine thymic environment

There is increasing evidence that human hematopoietic stem cells can develop into lymphocytes expressing T cell surface markers in the organ culture of murine embryonic thymic lobes. If human T cells with functional maturity are inducible from human stem cells in the mouse, it may be a useful model to investigate human T cell development and the human immune response in vivo. To approach this, we produced a hybrid cluster of murine fetal thymic epithelial cells and human cord blood-derived CD34(+) cells (hu/m cluster) using reaggregate thymic organ culture, and subsequently implanted it under the kidney capsule of NOD/SCID mice. The implanted hu/m cluster grew in volume under the kidney capsule and contained increased numbers of CD4(+)CD8(+)cells as well as CD4 or CD8 single-positive cells with low CD1a expression. These lymphocytes were also shown to possess activity for producing IL-2 and IL-4. Characteristics similar to human T cells also developed in the thymus of newly established mice lacking NK activity from NOD/SCID mice. These results indicate that functionally mature T cells can develop in vivo from human hematopoietic progenitors in the murine environment composed of thymic epithelial cells.     

Cotransplanted bone marrow derived mesenchymal stem cells (MSC) enhanced engraftment of hematopoietic stem cells in a MSC-dose dependent manner in NOD/SCID mice

Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34(+) -selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1x10(5) bone marrow derived human CD34(+) cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency.