Conditions that enable human hematopoietic stem cell engraftment in all NOD-SCID mice

BACKGROUND: Transplantation of human hematopoietic stem cells is the only true test of their long-term repopulation potential. Models are readily available to evaluate murine hematopoietic stem cells, but few exist that allow reliable quantification of human stem cells. The non-obese diabetic-severe combined immunodeficient (NOD-SCID) mouse model enables quantification of human hematopoietic stem cells, but the conditions that permit human engraftment in all animals have yet to be defined. The aims of the project were, therefore, to describe the variables that allow human engraftment in the NOD-SCID mouse model and the techniques that accurately quantify this engraftment. METHODS: NOD-SCID mice that had or had not received 250, 325, or 400 cGy irradiation received cord blood (CB) mononuclear or CD34+ cells i.v. or i.p. Mice were killed 6 weeks after transplantation, and the bone marrow, spleen, and thymus were harvested. Four-color flow cytometric analysis, semi-quantitative PCR, myeloid and erythroid progenitor, and stem cell assays were used to monitor human engraftment. RESULTS: A 250 or 325 cGy and i.v. injection of CB mononuclear or CD34+ cells is required to detect multilineage human engraftment in the bone marrow, spleen, or thymus of NOD-SCID mice. Four-color flow cytometric analysis and semi-quantitative PCR enable accurate detection of 0.1% human cells. Progenitor and stem cell assays provide functional information about the engrafted cells. CONCLUSIONS: Successful development of the NOD-SCID mouse model and techniques to assess human engraftment now allow it to be used reliably to analyze the effects of short-term cytokine exposure on the long-term repopulating capacity of CB stem cells.     

Successful multilineage engraftment of human cord blood cells in pigs after in utero transplantation

BACKGROUND: Successful engraftment of human hematopoietic stem and progenitor cells (HSPCs) in a large animal may serve not only as a model to study human hematopoiesis but also as a bioreactor to expand human HSPCs in vivo. The aim of this study was to accomplish xenotransplantation of human HSPCs into pig. METHODS: Total mononuclear or CD34-positive HSPCs obtained from human cord blood were xenotransplanted percutaneously under an ultrasonographic guidance into preimmune pig fetuses. Peripheral blood and bone marrow (BM) cells of recipient pigs were collected and analyzed for the presence of human cells by a polymerase chain reaction to detect human specific Alu sequence on DNA extracted from those cells. Fluorescence-activated cell sorting (FACS) analysis was also performed to detect human hematopoietic cells. RESULTS: Transplantation of human cord blood cells into pig fetuses aged less than 52 days postcoitus resulted in a good engraftment rate. In one case, engraftment was detected up to 315 days posttransplantation by polymerase chain reaction. Human hematopoietic cells were detectable also by FACS in peripheral blood and BM. Furthermore, human CD34+ HSPCs were also observed in the BM of recipients. Those CD34+ cells in BM were sorted by FACS and subjected to further analyses. First, in vitro colony formation assay resulted in formations of multilineage colonies. Second, when they were transplanted into an immunodeficient mouse they were engrafted in the mouse. CONCLUSIONS: These data indicate an engraftment of human HSPCs in pig BM. In utero transplantation of human HSPCs into a preimmune pig fetus is useful to establish a pig reproducing human hematopoiesis.     

人脐血造血干/祖细胞的磁力搅拌悬浮培养及移植实验

目的 探讨磁搅拌大规模培养体系对人脐血造血祖细胞的扩增效果以及扩增的人造血祖细胞植入动物体内后的造血重建情况.方法 从新鲜抗凝脐血中分离出单个核细胞(MNC),以添加干细胞因子、酪氨酸激酶受体3配基及血小板生成素的无血清培养体系进行培养.静态扩增组的细胞置于T25培养瓶中培养,磁搅拌悬浮扩增组(磁搅拌扩增组)的细胞采用Celstir装置进行培养,培养体系为50～100 ml.培养7 d后进行细胞计数、集落培养检测和细胞表面分子表达的测定.以不进行培养者为对照组.非肥胖糖尿病重症联合免疫缺陷(NOD/SCID)小鼠在接受2.5 Gy的亚致死剂量X射线照射后分别从尾静脉输入上述静态扩增组、磁搅拌扩增组和对照组的MNC(5×106个),另设不移植的空白对照组.观察小鼠的存活情况,6周后处死存活小鼠,检测骨髓细胞中CD34+细胞、CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 经过7天的培养,磁搅拌扩增组的造血祖细胞扩增倍数为(2.8±0.45)倍,明显高于静态扩增组的(2.1±0.48)倍(P<0.01).磁搅拌扩增组形成的红系集落、粒-巨噬细胞集落数均明显高于静态扩增组(P<0.05).静态扩增组扩增后的CD34+细胞、CD34+CD38-细胞和CD133+细胞含量均高于磁搅拌扩增组(P<0.05),而CD184+细胞和CD62L+细胞含量低于磁搅拌扩增组(P<0.01).移植后6周,对照组、静态扩增组和磁搅拌扩增组分别有3、4、5只小鼠存活,三组间两两比较,6周存活率的差异无统计学意义(P>0.05).存活6周的小鼠,其骨髓中能检人特异性CD34+细胞,以及CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞,也检测到人Alu基因和Cart-Ⅰ基因的表达.结论 磁搅拌培养能大规模扩增脐带血造血祖细胞,扩增的细胞能植入x射线照射的NOD/SCID小鼠,并重建其多系造血.     

Stable engraftment after megadose blood stem cell transplantation across the HLA barrier: the case for natural killer cells as graft-facilitating cells.

BACKGROUND: The case of a patient with chronic myelogenous leukemia who underwent transplantation with highly purified CD34+ peripheral blood stem cells from his two-antigen-mismatched mother is reported. No graft-versus-host disease has been observed so far and stable engraftment has been documented until day 100. METHODS: Weekly analysis of chimerism in different cellular subsets was performed using a quantitative polymerase chain reaction assay for nine short tandem repeat markers in leukocytes sorted by fluorescence-activated cell sorting. RESULTS: No donor CD4+ or CD8+ T cells have been detected up to 3 months after transplantation, whereas a rapid increase of donor CD56+ natural killer (NK) cells was observed in parallel with circulating donor CD34+ progenitors and myeloid cells. CONCLUSIONS: Because the graft contained virtually no T and NK cells, we believe the rapid in vivo generation of NK cells supported stable engraftment across the HLA barrier. The differentiation of CD34+ progenitors into NK cells might be a distinct feature of megadose stem cell transplants.     

Rapid establishment of long-term culture-initiating cells of donor origin after nonmyeloablative allogeneic hematopoietic stem-cell transplantation,and significant prognostic impact of donor T-cell chimerism on stable engraftment and progression-free survival

BACKGROUND: Nonmyeloablative allogeneic hematopoietic stem-cell transplantation (NST) allows establishment of donor hematopoiesis without eradication of recipient stem cells by chemoradiotherapy. Quantification of donor chimerism may predict graft failure and relapse. METHODS: We quantified donor long-term culture-initiating cells (LTC-IC) in nine patients during the early phase after NST and lineage-specific donor cells of myeloid (CD33+, CD34+, granulocytes) and lymphoid lineage (CD3+, CD4+, CD8+, CD56+) in 38 patients with a median follow-up of 40 weeks after NST. Conditioning therapy consisted of fludarabine 90 mg/m2 followed by total body irradiation of 2 Gy. RESULTS: Only rapid establishment of donor T-cell chimerism was essential for stable donor engraftment. Patients with less than 90% of donor T cells 4 weeks after NST had a significantly higher risk of relapse, graft rejection, or both (14 of 18 patients) than patients with donor T-cell chimerism of 90% and higher (3 of 20 patients). Although conditioning therapy was nonmyeloablative, a significant decrease of repopulating stem cells defined as LTC-IC was seen after 2 weeks followed by rapid recovery of LTC-IC to pretransplant values. Interestingly, all LTC-IC were from donor origin 2 and 4 weeks after NST, but rapid establishment of donor LTC-IC was not predictive for progression-free survival. CONCLUSIONS: Rapid establishment of lymphoid but not myeloid donor chimerism is a prognostic factor for stable donor engraftment after NST. It seems that an immunologic shield of alloreactive donor T cells is essential for early hematopoietic progenitors.     

Higher dose of CD34+ peripheral blood stem cells is associated with better survival after haploidentical stem cell transplantation in pediatric patients

Haploidentical stem cell transplantation (SCT) is increasingly used to treat pediatric patients with malignant or nonmalignant hematological disorders. The CD34+ dose of bone marrow or peripheral blood stem cells (PBSCs) has been shown to be an important determinant of the transplant outcome in adults under various preparative regimens. However, knowledge of the effect of the CD34+ dose in pediatric haploidentical SCT is limited. We analyzed the data of 348 pediatric patients (aged 2‐18 years) with acute or chronic leukemia, myelodysplastic syndrome (MDS), and other hematological disorders that received a transplant between 2002 and 2012. The results of multivariate analysis showed that PBSC CD34+ counts greater than 1.01 × 10⁶ kg^?1 improved platelet engraftment, improved overall survival, and reduced nonrelapse mortality. In contrast, a higher PBSC CD34+ dose did not affect the incidence of acute or chronic graft‐versus‐host disease, including engraftment syndrome. These data suggest that a PBSC CD34+ dose greater than 1.01 × 10⁶ kg^?1 is optimal for pediatric haploidentical SCT.     

Transfusion of peripheral blood stem cells from donor homozygous for a shared HLA-haplotype: avoiding fatal transfusion-associated graft-versus-host disease while preserving anti-leukemic effect

BACKGROUND: Fatal transfusion-associated graft-versus-host disease was observed in immunocompetent patients transfused with blood from donors homozygous for a shared haplotype with the recipient (the P-F1 barrier). We tested whether it was possible to carry out successful transplantation in a patient with relapsed acute myeloid leukemia, using peripheral blood stem cells from his HLA-homozygous brother (HLA A2, B46, DRB1 901) who shared a haplotype with the patient (HLA A2, B46,75, DRB1 901,12). METHODS: A CD34 positively selected cell fraction (5.46x 10(6) CD34 cells/kg) was infused first, followed by subsequent infusion of graded doses of donor T cells (total 7.25x10(7) T cells/kg). Nonmyeloablative chemotherapy with idarubicin and cytarabine was given during the transplantation to reduce the leukemic burden and facilitate engraftment. Polymerase chain reaction with the VNTR primers, D1S80, was used to detect engraftment. RESULTS: Complete remission (>300days) and successful donor engraftment (90%) were achieved. CONCLUSIONS: Peripheral blood stem cells transplantation from a donor with a homozygous shared haplotype is possible with a minimal preparative regimen.     

人脐血造血干/祖细胞的生物反应器大规模扩增及移植实验

目的 利用生物反应器大规模扩增人脐血造血干/祖细胞,并通过动物移植实验检验该方法的有效性.方法 采集抗凝脐血10份,分离出单个核细胞(MNC),分别进行生物反应器扩增培养和静态扩增培养.检测扩增前后细胞表面CD34、CD38、CD133、CD184和CD62L分子的表达,并进行造血干/祖细胞集落的培养.取非肥胖糖尿病重症联合免疫缺陷小鼠,以X射线照射后,分为4组,其中MNC组小鼠注射未经扩增培养的MNC;静态扩增组小鼠注射经过静态扩增培养的细胞;反应器扩增组小鼠注射经过生物反应器扩增培养的细胞;空白对照组小鼠注射生理盐水.移植后6周处死存活小鼠,收集骨髓细胞,检测其中CD45+、CD3+、CD19+和CD33+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 生物反应器扩增前MNC为(1.2～2.8)×108个,扩增后为(3.7～12.6)×108个,扩增后的细胞数明显高于静态扩增培养者(P<0.01).经生物反应器扩增后所形成的红系集落形成单位、粒-巨噬细胞集落形成单位数明显高于经静态扩增者(P<0.05).移植6周后,空白对照组小鼠均死亡,MNC组存活率为35%,静态扩增组存活率为30%,反应器扩增组存活率为62.9%,后者明显高于前二者(P<0.05).各组存活小鼠骨髓细胞中均检测到Alu基因和Cart-Ⅰ基因的表达以及人源CD33+、CD45+、CD3+及CD19+细胞.结论 利用生物反应器可大规模扩增人脐血造血干/祖细胞,所得细胞能植入小鼠体内,并能获得造血功能重建.     

Immune modulation to prevent antibody-mediated rejection after allogeneic hematopoietic stem cell transplantation

It has been shown that antibodies to donor CD34+/VEGFR-2+ stem cells or antibodies against mismatched HLA are associated with graft rejection after hematopoietic stem cell transplantation (HSCT). CD34/VEGFR-2 positive stem cells have been implicated to play a major role in engraftment after HSCT. In this study we treated four patients with an imminent risk of antibody-mediated rejection with immune modulation, i.e. plasma exchange, intravenous immunoglobulin (IVIG), and rituximab before HSCT. Three of the patients had been previously transplanted and rejected their initial grafts after 12 months, 1 month, and less than 1 month, respectively. The fourth patient was not transplanted previously but had HLA directed antibodies present against the graft. During the immune modulatory treatment we followed the pattern of antibodies in sera using FACS and microcytotoxicity assay. We could show that two patients had antibodies against donor CD34+/VEGFR-2+ cells while the other two had antibodies directed against HLA. All four patients tolerated the immune modulatory regimen without any side effects. In this preliminary study we show that immune modulatory treatment may be used to reduce antibody levels and to prevent rejection after HSCT. In two of the three patients which experienced previous rejections and had detectable anti-HLA or anti-CD34+/VEGFR-2+ antibodies, immune modulation resulted in engraftment. In the fourth patient with known anti-HLA-class I antibodies, the treatment also resulted in engraftment. Our results encourage further studies regarding this treatment regimen.     

由脐血单个核细胞和富集的CD34+细胞扩增的造血干/祖细胞的生理功能比较

目的 探讨由脐血单个核细胞(MNC)和富集的CD34+细胞起始扩增所得的造血干/祖细胞在体内植入及造血重建的能力.方法 从人脐血中分离出MNC和CD34+细胞,在体外扩增7 d.将非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠分为四组,在接受亚致死剂量铯源照射后,进行细胞移植,实验A组接受由MNC培养得到的CD34+细胞和CD34-细胞;实验B组接受由富集的CD34+细胞培养得到的CD34+细胞和CD34-细胞;阳性对照组接受从脐血新鲜分离的CD34+细胞和CD34-细胞;阴性对照组不接受细胞移植,仅输注相同体积的IMDM培养基.6周后处死存活的小鼠,取其骨髓、脾脏和外周血细胞,分别进行细胞表型分析、集落和人特异性基因的检测.结果 经过体外扩增,以富集的CD34+细胞起始培养者的细胞总扩增倍数为39.8倍,远高于以MNC为起始细胞者的1.88倍.移植6周后,所有接受细胞移植的小鼠均存活,存活小鼠的骨髓和脾脏细胞中均能检测到人源细胞(CD45+细胞)及人源的各系血细胞,实验A组各类细胞的含量稍高于实验B组,且接受细胞移植小鼠的骨髓和脾脏细胞中可检测出人特异的Alu序列.结论 与从脐血中新鲜分离的细胞相比,扩增后的造血干/祖细胞的体内植入能力有所下降,以MNC起始扩增的造血干/祖细胞体内植入能力优于以富集的CD34+细胞起始扩增者,但二者体内造血重建能力的差异不显著.     

Successful generation of donor specific hematopoietic stem cell lines from co-cultured bone marrow with human embryonic stem cell line: a new methodology

We report the generation of 30 healthy human embryonic stem cell (h-ESC) lines from 33 voluntary oocyte donors using a donor somatic cell nuclear transfer (SCNT) technique on 190 oocytes. Our aim was to coculture them with their own bone marrow (BM) to generate hematopoietic progenitor cells for therapeutic purposes. Pluripotency and undifferentiated stage were confirmed using molecular cell surface markers. Normal karyotype of these cell lines was confirmed. Here we demonstrate that SCNT-h-ESCs differentiate to hematopoietic precursors when cocultured with unmodified, nonirradiated donor BM. We did not use any xenogeneic material for this hematopoietic differentiation. Hematopoietic precursors derived from them expressed cell surface antigens CD45/34. When further cultured with hematopoietic growth factors these hematopoietic precursors formed characteristic myeloid, erythroid, and megakaryocyte lineages. Phenotypic CD34+ cells derived from NT-h-ESCs were functionally similar to their counterparts in primary hematopoietic tissues like BM, umbilical cord, and blood. More terminally differentiated hematopoietic cells derived from h-ESCs under these culture conditions also expressed normal surface antigens like glycophorin A on erythroid cells, CD15 on myeloid cells, and CD41 on megakaryocytes. We report generation of hematopoietic progenitor cells from h-ESC lines by a SCNT technique, with differentiation into further lineages with structural and functional similarities to their adult counterparts in vivo. This novel alternative source of CD34+ stem cells from h-ESC lines generated without any xenogeneic material might be used to create transplantation tolerance, to implement regenerative medicine, and to treat autoimmune disorders.     

The effect of human fetal liver-derived mesenchymal stem cells on CD34+ hematopoietic stem cell repopulation in NOD/Shi-scid/IL-2Rã(null) mice

Mesenchymal stem cells (MSCs) are progenitors that are capable of differentiating into mesenchymal tissues. They are known to support allogeneic hematopoietic stem cell transplantation by facilitating engraftment without increasing the risk of graft-versus-host disease. We optimized culture conditions for human fetal liver-derived MSCs (hFL-MSCs) to investigate the role of hFL-MSCs on repopulation of hematopoietic stem cells in NOD/Shi-scid/IL-2Rγ(null) (NOG) mice using CD34(+) hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB). FL-MSCs and CD34(+) HSCs were prepared from fetal liver and UCB, respectively. Twenty-four hours after irradiation, CD34(+) HSCs and hFL-MSCs were injected intravenously and intratibially into NOG mice. During 24 weeks posttransplantation, engraftment levels of human cells were analyzed in bone marrow, peripheral blood, and spleen of transplanted mice by flow cytometry. hFL-MSCs showed a fibroblast-like morphology and immunophenotypic characteristics appropriate for MSCs. hFL-MSCs prolonged the survival of NOG mice that had been cotransplanted with UCB CD34(+) cells. Fluorescence-activated cell-sorting analysis showed that engraftment of human cells was increased by cotransplantation of hFL-MSCs. However, significant enhancement of human cell engraftment was not detected in NOG mice regardless of the number of cotransplanted MSCs. Although survival of repopulating NOG mice and engraftment of human cells were prolonged by cotransplantation of hFL-MSCs, 8.0 × 10(6) MSCs were not sufficient to increase HSC engraftment in irradiated NOG mice in vivo.     

Engraftment of discordant xenogeneic swine bone marrow cells in immunodeficient mice

Abstract: We have recently demonstrated that swine bone marrow cells (BMC) can engraft in C.B-17 scid mice. While engraftment is enhanced by providing donor-specific porcine cytokines, the level of swine hematopoiesis declines between 3 and 6 weeks post-transplant. In the present study, the utility of several strains of immunodeficient mice as recipients of swine hematopoietic cells has been determined by comparing levels of swine bone marrow engraftment at 3 weeks after bone marrow transplant. Irradiated recipients were injected with lxlO8 swine BMC and were treated daily with porcine cytokines. The presence of swine cells in BMT recipients was detected by flow cytometry and marrow colony-forming assays. Recombination activating gene-1 (RAG-1)-deficient mice were not permissive for the engraftment of swine BMC, even with administration of increased doses of whole body irradiation, or with depleting anti-NK cell antibody. In contrast, NOD-scid mice showed improved swine BMC engraftment compared to C.B-17 scid mice. Levels of swine class I+, myeloid, and CD2+ cells in bone marrow, spleen, and peripheral blood, and the number of porcine myeloid progenitor cells was significantly higher in NOD-scid recipients than in simultaneous C.B-17 scid recipients. In addition, the sera from C.B-17 scid mice markedly inhibited the proliferation of swine BMC in vitro. A weaker inhibitory effect was also mediated by sera from RAG-1-deficient mice, but not by sera from NOD-scid mice. Together, our results indicate that multiple host elements resist xenogeneic hematopoietic engraftment and function, some of which are clearly independent of host T, B, or NK cells. Understanding the basis for the advantage of NODscid mice as recipients of discordant xenogeneic porcine BMC will help to identify these elements.     

CD34+ selected bone marrow grafts are radioprotective and establish mixed chimerism in dogs given high dose total body irradiation.

BACKGROUND: Canine stem cell transplantation models have provided important preclinical information for human clinical studies. The recent cloning of cDNA for canine CD34 and the production of monoclonal antibodies that recognize canine CD34 have been the basis for the development of techniques for the large-scale enrichment of canine hematopoietic progenitor cells. In this study, we evaluated the in vivo functional properties of canine bone marrow CD34+ cells after a myeloablative conditioning regimen. METHODS: After 920 cGy total body irradiation, three dogs received infusion of autologous CD34+ selected cells from the marrow, three dogs CD34+ depleted autologous marrow cells, and two dogs received CD34+ autologous marrow cells that were immunomagnetically selected and then further purified by cell sorting. In addition, four dogs received allogeneic marrow enriched for CD34+ cells from dog leukocyte antigen-identical littermates to investigate long-term repopulating function of CD34+ cells. Chimerism studies were performed using polymerase chain reaction to detect highly polymorphic microsatellite markers. RESULTS: In three recipients of autologous marrow enriched for CD34+ cells to between 29% and 70% (1.6 x 10(6) to 3.4x10(6) CD34+ cells/kg), prompt and full hematopoietic recovery occurred, whereas in three dogs that received marrow depleted of CD34+ cells (1 x 10(7) cells/kg), no hematopoietic recovery was achieved. In two dogs that received highly purified CD34+ cells (purity: 98% and 96%, 0.79x10(6) to 0.547x 10(6) CD34+ cells/kg), delayed but full hematopoietic recovery was seen. Three of four allograft recipients of 1.75x10(6) to 6.8x10(6) CD34+ cells/kg engrafted and showed full hematopoietic recovery, whereas one dog rejected the graft. The three long-term survivors showed stable mixed hematopoietic chimerism with predominantly donor hematopoiesis. CONCLUSION: Transplantation of canine CD34+ cells after lethal total body irradiation provides radioprotection and gives rise to long-term hematopoietic reconstitution. Stable donor/host mixed chimerism was observed in allograft recipients most likely as a result of T-cell depletion of the grafts. Our findings suggest a future role for canine preclinical transplant studies involving in vitro manipulation of hematopoietic pro.     

Ex vivo expansion and transplantation of hematopoietic stem/progenitor cells supported by mesenchymal stem cells from human umbilical cord blood

Human mesenchymal stem cells (MSCs) are multipotential and are detected in bone marrow (BM), adipose tissue, placenta, and umbilical cord blood (UCB). In this study, we examined the ability of UCB-derived MSCs (UCB-MSCs) to support ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs) from UCB and the engraftment of expanded HSPCs in NOD/SCID mice. The result showed that UCB-MSCs supported the proliferation and differentiation of CD34+ cells in vitro. The number of expanded total nucleated cells (TNCs) in MSC-based culture was twofold higher than cultures without MSC (control cultures). UCB-MSCs increased the expansion capabilities of CD34+ cells, long-term culture-initiating cells (LTC-ICs), granulocyte-macrophage colony-forming cells (GM-CFCs), and high proliferative potential colony-forming cells (HPP-CFCs) compared to control cultures. The expanded HSPCs were transplanted into lethally irradiated NOD/SCID mice to assess the effects of expanded cells on hematopoietic recovery. The number of white blood cells (WBCs) in the peripheral blood of mice transplanted with expanded cells from both the MSC-based and control cultures returned to pretreatment levels at day 25 posttransplant and then decreased. The WBC levels returned to pretreatment levels again at days 45-55 posttransplant. The level of human CD45+ cell engraftment in primary recipients transplanted with expanded cells from the MSC-based cultures was significantly higher than recipients transplanted with cells from the control cultures. Serial transplantation demonstrated that the expanded cells could establish long-term engraftment of hematopoietic cells. UCB-MSCs similar to those derived from adult bone marrow may provide novel targets for cellular and gene therapy.     

Porcine hematopoiesis on primate stroma in long-term cultures: enhanced growth with neutralizing tumor necrosis factor-alpha and tumor growth factor-beta antibodies

BACKGROUND: Donor hematopoiesis is at a competitive disadvantage when bone marrow transplantation is across species barriers. This could present major limitations to xenogeneic stem cell transplantation as an approach to tolerance induction. An in vitro model of xenogeneic engraftment was established to identify inhibitors of porcine hematopoiesis in a primate environment. METHODS: Porcine bone marrow cells (BMC), in the presence or absence of primate CD34+ positive cells, were cultured for 4-6 weeks on primate stroma with porcine cytokines. Cellularity and growth of colony-forming cells were indicators of hematopoietic growth. Effects of soluble factors were determined by using Transwell inserts to separate porcine cells from stroma. Neutralizing antibodies for human transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) were added to cultures. RESULTS: Porcine hematopoiesis can be maintained in long-term cultures on primate stroma with pig cytokines. Adding BMC to the stroma below Transwell-containing porcine cells dramatically inhibited porcine hematopoiesis, showing an inhibitory role for soluble factors. Neutralizing antibodies against TNF-alpha or TGF-beta caused a modest enhancement of porcine hematopoiesis; however, the combination of both led to a substantial increase. Inhibitory effects of these cytokines were confirmed by adding TNF-alpha and TGF-beta to porcine cultures. CONCLUSIONS: Porcine cells may be more sensitive to inhibitory effects of TNF-alpha and TGF-beta than primate cells and are at a disadvantage when in a primate environment. Potential implications of this observation are discussed in the context of establishing specific immune tolerance via mixed chimerism to facilitate xenotransplantation.     

Gene delivery using human cord blood-derived CD34+cells into inflamed glomeruli in NOD/SCID mice

BACKGROUND: Bone marrow reconstitution using genetically-modified hematopoietic stem cells has been reported to confer resistance to inflammation and prevent renal injury in glomerulonephritis. Although this strategy has potentials for clinical use, taking hematopoietic stem cells from bone marrow is highly stressful for patients. In this regard, umbilical cord blood may be a useful alternative and, therefore, we focused on their suitability as a source of hematopoietic stem cells for transplantation-based therapy for glomerulonephritis. METHODS: CD34+ cells were obtained from human umbilical cord blood, retrovirally transduced with human beta-glucuronidase (HBG) gene, and transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After confirming the successful chimerism, these mice were treated with lipopolysaccharide (LPS), and local HBG expression in glomeruli was examined using immunohistochemical analysis, HBG bioassay, and Western blot analysis. RESULTS: Clonogenic assay showed that 88.4 +/- 5.9% burst-forming unit-erythroid (BFU-E), 79.7 +/- 11.4% in colony-forming unit-macrophage (CFU-M), and 81.1 +/- 14.1% in colony-forming unit-granulocyte (CFU-G), respectively, possessed the transgene after transfection, suggesting that precommited cells were susceptible to retroviral infection. Flow cytometric analysis revealed that 24.1 +/- 14.5% of bone marrow cells in these chimera mice expressed human lymphocyte antigen (HLA) 8 weeks after transplantation. Also, clonogenic assay showed that a sustained engraftment of human hematopoietic cells expressed HBG. CD14-positive cells were recruited into the glomeruli upon LPS treatment and they secreted bioactive HBG, suggesting that cord blood-derived CD34+cells may differentiate into monocyte lineage while maintaining the expression of the transgene. CONCLUSION: These data indicate that umbilical cord blood cells can be utilized as a source of hematopoietic stem cells for the transplantation-based therapy of glomerulonephritis.     

Transplantation of human peripheral blood stem cells into fetal rhesus monkeys (Macaca mulatta)

BACKGROUND: Methods for assessing engraftment efficiency have been explored in a primate xenogeneic model of in utero hematopoietic stem cell transplantation. METHODS: Human peripheral blood stem cells (PBSC) were obtained by leukapheresis from a human male donor after 4 days of administration of recombinant human granulocyte-colony stimulating factor (5 microg/kg/ day). PBSC were enriched for the CD34+ population with and without T-cell depletion. The resulting mononuclear cells consisted of two cell populations, one that was stem cell enriched (0.83% CD3+ cells, 95% CD34+; group 1) and one that was stem cell enriched and T-cell depleted (<0.03% CD3+ cells, 98% CD34+; group 2). Four fetal monkeys (two per group) received either two or four i.p. injections (approximately 5x10(6) cells/injection) via ultrasound guidance every other day over a 7-day period (gestational days 50, 52, 54, and 56). One fetus in each group also received i.p. recombinant human stem cell factor (25 microg/kg) and recombinant human granulocyte-colony stimulating factor (10 microg/kg) posttransplant every 10 days from gestational day 60-150. RESULTS: Four healthy newborns were delivered at term, and specimens were analyzed by polymerase chain reaction for the human Y chromosome (birth, monthly to 6 months; blood, marrow, progenitor assays). Polymerase chain reaction results were positive for all four newborns in all specimens assessed, and flow cytometric analysis for human CD45 in marrow showed engraftment ranging from 0.1-1.7%. There was no evidence of graft-versus-host disease in any of the animals. CONCLUSION: These studies show that (1) multilineage engraftment of human PBSC can be achieved in the fetal rhesus recipient, (2) the rhesus fetus appears to tolerate relatively high numbers of human CD3+ cells, and (3) healthy chimeric rhesus infants can be delivered at term after multiple in utero procedures.     

Intramyocardial CD34+ cell transplantation combined with off-pump coronary artery bypass grafting

This report describes a new therapeutic approach for severe ischemic heart disease, intramyocardial transplantation of autologous bone marrow-derived CD34 + cells combined with off-pump coronary artery bypass grafting (CABG). CD34 is widely known as a cell surface antigen expressed on hematopoietic stem cells, and recent experimental studies have shown that CD34 + cells include endothelial progenitor cells. We used the Isolex 300i magnetic cell selection system to separate CD34 + cells from bone marrow cells. This report describes the first case treated with the combination of off-pump CABG and cell transplantation for therapeutic angiogenesis and myocardial regeneration. The transplantation of autologous bone marrow-derived CD34 + cells improved perfusion of the ungraftable ischemic area.