Transplantation of endothelial cells corrects the phenotype in hemophilia A mice

BACKGROUND: The deficiency of factor VIII, a co-factor in the intrinsic coagulation pathway results in hemophilia A. Although FVIII is synthesized largely in the liver, the specific liver cell type(s) responsible for FVIII production is controversial. OBJECTIVE: This study aimed to determine the cellular origin of FVIII synthesis and release in mouse models. METHODS: We transplanted cells into the peritoneal cavity of hemophilia A knockout mice. Plasma FVIII activity was measured using a Chromogenix assay 2-7 days after cell transplantation, and phenotypic correction was determined with tail-clip challenge 7 days following cell transplantation. Transplanted cells were identified by histologic and molecular assays. RESULTS: Untreated hemophilia A mice, as well as mice treated with the hepatocyte-enriched fraction, showed extensive mortality following tail-clip challenge. In contrast, recipients of unfractionated liver cells (mixture of hepatocytes, liver sinusoidal endothelial cells (LSEC), Kupffer cells, and hepatic stellate cells) or of the cell fraction enriched in LSECs survived tail-clip challenge (P < 0.001). FVIII was secreted in the blood stream in recipients of unfractionated liver cells, LSECs and pancreatic islet-derived MILE SVEN 1 (MS1) endothelial cells. Although transplanted hepatocytes maintained functional integrity in the peritoneal cavity, these cells did not produce detectable plasma FVIII activity. CONCLUSIONS: The assay of cell transplantation in the peritoneal cavity showed that endothelial cells but not hepatocytes produced phenotypic correction in hemophilia A mice. Therefore, endothelial cells should be suitable additional targets for cell and gene therapy in hemophilia A.     

异种肝前体细胞移植治疗急性肝损伤大鼠

背景：成体肝前体细胞可在受体肝脏内定植并分化为肝细胞。不过,异种肝前体细胞移植能否促进急性肝损伤的恢复,脾脏微环境能否促进移植物的存活和向肝细胞分化,尚没有研究。目的：评价异种肝前体细胞移植治疗急性肝损伤的作用;监测移植肝前体细胞在大鼠脾脏实质内的定植及向肝细胞的分化。方法：体外培养雄性小鼠来源的肝前体细胞系肝上皮样前体细胞。通过CCl4腹腔注射联合2/3肝切除构建急性肝损伤大鼠模型,进行肝上皮样前体细胞脾脏移植。在肝切除后1,5,14和21 d,苏木精-伊红染色观察肝脏病理改变,全自动生化分析仪监测血清转氨酶变化,PCR反应检测脾脏组织Y染色体特异性序列Sry,脾脏CK-19和Alb免疫组织化学追踪移植肝上皮样前体细胞的植入和肝细胞分化。结果与结论：肝上皮样前体细胞可在体外长期培养,保持增殖能力和双向分化潜能。肝上皮样前体细胞脾脏移植后,肝损伤大鼠肝细胞肿胀明显减轻,丙氨酸转氨酶和天门冬氨酸转氨酶下降更明显。移植后1,5,14和21 d,脾脏DNA中均能检测到Sry序列。在整个实验期间CK-19阳性细胞在大鼠脾脏实质内始终存在。Alb阳性细胞在移植后5 d在脾脏实质中出现,随后阳性细胞数逐渐增多。实验表明,移植肝前体细胞能在大鼠脾脏实质中植入,并分化为肝细胞,能有效促进CCl4腹腔注射联合2/3肝切除诱导的大鼠急性肝损伤的修复过程。     

Correction of murine hemophilia A by hematopoietic stem cell gene therapy.

A serious complication of current protein replacement therapy for hemophilia A patients with coagulation factor VIII (FVIII) deficiency is the frequent development of anti-FVIII inhibitor antibodies that preclude therapeutic benefit from further treatment. Induction of tolerance by persistent high-level FVIII synthesis following transplantation with hematopoietic stem cells expressing a retrovirally delivered FVIII transgene offers the possibility of permanently correcting the disease. Here, we transplanted bone marrow cells transduced with an optimized MSCV-based FVIII oncoretroviral vector into immunocompetent hemophilia A mice that had been conditioned with a potentially lethal dose of irradiation (800 cGy), a sublethal dose of irradiation (550 cGy), or a nonmyeloablative preparative regimen involving busulfan. Therapeutic levels of FVIII (42, 18, and 11% of normal, respectively) were detected in the plasma of the transplant recipients for the duration of the study (over 6 months). Moreover, subsequent challenge with recombinant FVIII elicited at most a minor anti-FVIII inhibitor antibody response in any of the experimental animals, in contrast to the vigorous neutralizing humoral reaction to FVIII that was stimulated in naive hemophilia A mice. These findings represent an encouraging advance toward potential clinical application and long-term amelioration or cure of this progressively debilitating, life-threatening bleeding disorder.     

Intra‐articular injection of mesenchymal stem cells expressing coagulation factor ameliorates hemophilic arthropathy in factor VIII‐deficient mice

Summary. Background: Transplantation of cells overexpressing a target protein represents a viable gene therapeutic approach for treating hemophilia. Here, we focused on the use of autologous mesenchymal stem cells (MSCs) expressing coagulation factor for the treatment of coagulation factor VIII (FVIII) deficiency in mice. Methods and Results: Analysis of luciferase gene constructs driven by different promoters revealed that the plasminogen activator inhibitor‐1 (PAI‐1) gene promoter coupled with the cytomegalovirus promoter enhancer region was one of the most effective promoters for producing the target protein. MSCs transduced with the simian immunodeficiency virus (SIV) vector containing the FVIII gene driven by the PAI‐1 promoter expressed FVIII for several months, and this expression was maintained after multiple mesenchymal lineage differentiation. Although intravenous injection of cell supernatant derived from MSCs transduced with an SIV vector containing the FVIII gene driven by the PAI‐1 promoter significantly increased plasma FVIII levels, subcutaneous implantation of the MSCs resulted in a transient and weak increase in plasma FVIII levels in FVIII‐deficient mice. Interestingly, intra‐articular injection of the transduced MSCs significantly ameliorated the hemarthrosis and hemophilic arthropathy induced by knee joint needle puncture in FVIII‐deficient mice. The therapeutic effects of a single intra‐articular injection of transduced MSCs to inhibit joint bleeding persisted for at least 8 weeks after administration. Conclusions: MSCs provide a promising autologous cell source for the production of coagulation factor. Intra‐articular injection of MSCs expressing coagulation factor may offer an attractive treatment approach for hemophilic arthropathy.     

Graft failure after allogeneic hematopoietic stem cell transplantation

Graft failure is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) defined as either lack of initial engraftment of donor cells (primary graft failure) or loss of donor cells after initial engraftment (secondary graft failure). Successful transplantation depends on the formation of engrafment, in which donor cells are integrated into the recipient’s cell population.In this paper, we distinguish two different entities, graft failure (GF) and poor graft function (PGF), and review the current comprehensions of the interactions between the immune and hematopoietic compartments in these conditions. Factors associated with graft failure include histocompatibility locus antigen (HLA)-mismatched grafts, underlying disease, type of conditioning regimen and stem cell source employed, low stem cell dose, ex vivo T-cell depletion, major ABO incompatibility, female donor grafts for male recipients, disease status at transplantation.Although several approaches have been developed which aimed to prevent graft rejection, establish successful engraftment and treat graft failure, GF remains a major obstacle to the success of allo-HSCT.Allogeneic hematopoietic stem cell transplantation (allo-HSCT) still remains to be the curative treatment option for various non-malignant and malignant hematopoietic diseases. The outcome of allo-HSCT primarily depends on the engraftment of the graft. Graft failure (GF), is a life-threatening complication which needs the preferential therapeutic manipulation. In this paper, we focused on the definitions of graft failure / poor graft function and also we reviewed the current understanding of the pathophysiology, risk factors and treatment approaches for these entities.     

Three‐dimensional functional human myocardial tissues fabricated from induced pluripotent stem cells

The most radical treatment currently available for severe heart failure is heart transplantation; however, the number of donor hearts is limited. A better approach is to make human cardiac tissues. We developed an original cell sheet‐based tissue‐engineering technology to fabricate human cardiac tissue by layering myocardial cell sheets. Human induced pluripotent stem (iPS) cells were differentiated into cardiomyocytes to fabricate cardiomyocyte sheets. Initially, three‐layer human iPS cardiomyocyte (hiPSCM) sheets were transplanted on subcutaneous tissues of nude rats. Next, to fabricate thicker tissue, three‐layer sheets were transplanted on one day, then additional three‐layer sheets were transplanted onto them the following day, after the first sheets were vascularized. On day 3, the final three‐layer sheets were again transplanted, creating a nine‐layer graft (multi‐step transplantation procedure). In the last step, six‐layer sheets were transplanted on fat tissues of the inguinal portion, which were subsequently resected together with the femoral arteries and veins to make transplantable grafts with connectable vessels. They were then transplanted ectopically to the neck portion of other rats by anastomosing vessels with the host's jugular arteries and veins. Transplanted three‐layer hiPSCMs were beating and, histologically, showed a cardiac muscle‐like structure with vascular systems. Moreover, transplanted hiPSCMs proliferated and matured in vivo. Significantly thicker tissues were fabricated by a multi‐step transplantation procedure. The ectopically transplanted graft survived and continued to beat. We succeeded in fabricating functional human cardiac tissue with cell sheet technology. Transplanting this cardiac tissue may become a new treatment option for severe heart failure. Copyright © 2015 John Wiley & Sons, Ltd.     

Lack of desmopressin (DDAVP) response in men with hemophilia A following liver transplantation

Summary.  Although hemophilia A, a congenital disorder caused by defective or deficient factor VIII:C (FVIII), is cured by liver transplantation, the exact site of hepatic FVIII production is unknown. Further, while intracellular co-localization of FVIII and von Willebrand factor (VWF) is required for in vitro FVIII secretion, whether it is required for in vivo FVIII secretion is not known. An ideal setting to study this problem is in individuals with hemophilia A following liver transplantation, as their FVIII is synthesized primarily in hepatic, but not extrahepatic endothelial cells, while VWF is synthesized primarily in extrahepatic vascular endothelium. Following liver transplantation for end-stage liver disease, three hemophilic men showed VWF, but no FVIII response to (DDAVP) infusion. By contrast, both VWF and FVIII increased in a non-hemophilic transplant recipient after DDAVP. These findings support a model in which intracellular co-localization of FVIII and VWF is necessary for in vivo FVIII secretion after DDAVP.     

A novel method to align cells in a cardiac tissue‐like construct fabricated by cell sheet‐based tissue engineering

Fabrication of cardiac tissue from human induced pluripotent stem cell‐derived cardiomyocytes (hiPS‐CMs) has received great interest, but a major challenge facing researchers is the alignment of cardiomyocytes in the same direction to optimize force generation. We have developed a novel method of fabricating a cardiac tissue‐like construct with aligned cells based on the unidirectional stretching of an hiPS‐CM sheet. A square cell sheet was harvested from a temperature‐responsive culture dish and placed on a silicone surface, and an extending force was imposed on the silicone to stretch the cell sheet along one direction. To enable evaluation of cardiomyocyte morphology in vitro, a cell sheet was constructed by coculture of hiPS‐CMs and human adipose‐derived stem cells. In separate experiments, a stretched double‐layered cell sheet constructed from hiPS‐CMs alone was transplanted onto the muscle of an athymic rat, and its features were compared with those of a nonstretched (control) cell sheet. Immediately after stretching, the stretched cell sheet was significantly longer than the control cell sheet. Immunohistological analysis revealed that the cardiomyocytes showed unidirectional alignment in the stretched cell sheet but random directionality in the control cell sheet. Two weeks after transplantation, immunohistology demonstrated that the stretched cell sheet had retained the unidirectionality of its myocardial fibers and had an orientation intensity that was higher than that of the control cell sheet after transplantation or the stretched cell sheet before transplantation. Our technique provides a simple method of aligning an hiPS‐CM‐derived cardiac tissue‐like construct without the use of a scaffold.     

Stem Cell‐Based Therapies for Ischemic Stroke: Preclinical Results and the Potential of Imaging‐Assisted Evaluation of Donor Cell Fate and Mechanisms of Brain Regeneration

Stroke is the second most common cause of death and is a major cause of permanent disability. Given the current demographic trend of an ageing population and associated increased risk, the prevalence of and socioeconomic burden caused by stroke will continue to rise. Current therapies are unable to sufficiently ameliorate the disease outcome and are not applicable to all patients. Therefore, strategies such as cell‐based therapies with mesenchymal stem cell (MSC) or induced pluripotent stem cell (iPSC) pave the way for new treatment options for stroke. These cells showed great preclinical promise despite the fact that the precise mechanism of action and the optimal administration route are unknown. To gain dynamic insights into the underlying repair processes after stem cell engraftment, noninvasive imaging modalities were developed to provide detailed spatial and functional information on the donor cell fate and host microenvironment. This review will focus on MSCs and iPSCs as types of widely used stem cell sources in current (bio)medical research and compare their efficacy and potential to ameliorate the disease outcome in animal stroke models. In addition, novel noninvasive imaging strategies allowing temporospatial in vivo tracking of transplanted cells and coinciding evaluation of neuronal repair following stroke will be discussed.     

Engineering tissues, organs and cells

Patients suffering from diseased and injured organs may be treated with transplanted organs; however, there is a severe shortage of donor organs that is worsening yearly, given the ageing population. In the field of regenerative medicine and tissue engineering, scientists apply the principles of cell transplantation, materials science and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Therapeutic cloning, where the nucleus from a donor cell is transferred into an enucleated oocyte in order to extract pluripotent embryonic stem cells, offers a potentially limitless source of cells for tissue engineering applications. The stem cell field is also advancing rapidly, opening new options for therapy, including the use of amniotic and placental fetal stem cells. This review covers recent advances that have occurred in regenerative medicine and describes applications of these technologies using chemical compounds that may offer novel therapies for patients with end-stage organ failure.     

Successful immune tolerance induction by FVIII in hemophilia A patients with inhibitor may occur without deletion of FVIII‐specific T cells

Summary. Background: The development of an inhibitor is the major complication facing patients with hemophilia A treated by administration of factor (F) VIII concentrates. Restoration of tolerance to FVIII can be achieved by prolonged administration of FVIII (immune tolerance induction, ITI). Although ITI has been used for more than 30 years in patients with hemophilia A and inhibitor, its mechanism of action is still poorly understood. Objectives: As administration of high doses of antigen can induce the apoptosis of the T cells recognizing the antigen, a potential mechanism of action of ITI may be the deletion of FVIII‐specific T cells. Patients/Methods: We studied the CD4+ T‐cell response to FVIII in five (one mild, one moderate and three severe) patients successfully desensitized by administration of FVIII and in control subjects. Results: Following repeated stimulation with autologous dendritic cells loaded with FVIII, FVIII‐specific T oligoclonal cell lines were expanded from the blood of one of the successfully desensitized patients. The FVIII‐specific T cells produced IL‐5, IL‐13 and IL‐2. By contrast, FVIII‐specific T‐cell lines could not be derived from three patients with mild hemophilia A without inhibitor or from four normal control subjects. Conclusions: These data represent the first analysis of the cellular mechanisms regulating the induction of tolerance to FVIII. They demonstrate that successful tolerance induction may occur without deletion of FVIII‐specific T cells.     

Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice

Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type–specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain–deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.     

非清髓异基因外周血造血干细胞移植治疗血液病的临床观察

目的　探讨非清髓异基因外周血造血干细胞移植 (NASCT)在治疗血液病中的意义。方法　采用NASCT治疗 33例HLA相合的血液病患者。男 2 0例 ,女 13例 ,中位年龄 36岁 (18～ 5 9岁 )。33例中急性白血病第 1次完全缓解期 (CR1 ) 11例 ,CR2～ 34例 ,难治复发性急性白血病未缓解期 3例 ,重型再生障碍性贫血 (SAA) 4例 ,慢性粒细胞白血病慢性期 7例 ,骨髓增生异常综合征 (MDS) 2例 ,慢性淋巴细胞白血病和骨髓纤维化各 1例。非清髓预处理方案 :白血病患者采用环磷酰胺 (CTX)、阿糖胞苷及CD3单克隆抗体 ,6例患者在此基础上加用氟达拉宾。SAA和MDS患者采用CTX和抗胸腺细胞球蛋白。结果　 33例均顺利渡过造血抑制期。平均移植后第 10 .5天 (移植后第 8～ 2 1天 )中性粒细胞计数 >0 .5× 10 9 L ,第 15天 (移植后第 10～ 30天 )血小板计数 >30× 10 9 L。 33例中供者细胞完全植入2 4例 (其中 13例于移植后 1～ 6个月由供受者嵌合性植入转为完全植入 ) ,稳定混合嵌合体 4例 ,移植排斥 5例。 33例中发生急性和慢性移植物抗宿主病各 7例 (2 1.2 % )。随诊 2～ 36个月 2 5例 (75 8% )仍存活。结论　NASCT简便安全 ,并发症少 ,疗效较好 ,为治愈血液病提供了新手段。     

Scaffolding kidney organoids on silk

End stage kidney disease affects hundreds of thousands of patients in the United States. The therapy of choice is kidney replacement, but availability of organs is limited, and alternative sources of tissue are needed. Generation of new kidney tissue in the laboratory has been made possible through pluripotent cell reprogramming and directed differentiation. In current procedures, aggregates of cells known as organoids are grown either submerged or at the air‐liquid interface. These studies have demonstrated that kidney tissue can be generated from pluripotent stem cells, but they also identify limitations. The first is that perfusion of cell aggregates is limited, restricting the size to which they can be grown. The second is that aggregates lack the structural integrity required for convenient engraftment and suturing or adhesion to regions of kidney injury. In this study, we evaluated the capacity of silk to serve as a support for the growth and differentiation of kidney tissue from primary cells and from human induced pluripotent stem cells. We find that cells can differentiate to epithelia characteristic of the developing kidney on this material and that these structures are maintained following engraftment under the capsule of the adult kidney. Blood vessel investment can be promoted by the addition of vascular endothelial growth factor to the scaffold, but the proliferation of stromal cells within the graft presents a challenge, which will require some readjustment of cell growth and differentiation conditions. In summary, we find that silk can be used to support growth of stem cell derived kidney tissue.     

Co-combination of islets with bone marrow mesenchymal stem cells promotes angiogenesis

BackgroundIslet transplantation is a commonly therapeutic strategy for diabetes mellitus. However, avascular phase and the poor formation of blood vessels in the late period lead to islet allograft loss which contributed to inefficiency and short-acting of islet transplantation. Recently, to speed up new angiogenesis and increase the density of blood vessels around transplanted islets became the hotspot in research of islet transplantation.MethodsIn this study, we undergone co-combination transplantation of allogeneic islet and bone marrow mesenchymal stem cells (BM-MSCs) into non-obese diabetic (NOD) mice and investigated the influence of BM-MSCs in transplanted islet function and neovascularization.ResultsIn mice of co-combination transplantation of islet with BM-MSCs, level of blood glucose was improved compared with only BM-MSCs transplanted mice; proliferation of islet cell was enhanced while apoptosis of islet cell was reduced; 2, 4, and 8 weeks post transplantation, peripheral vascular density of islet grafts were significantly more than the islet transplantation group alone; donor lymphocytic chimerism in graft was increased. In result of immunofluorescence analysis, we observed that BM-MSCs can migrate to transplanted islet, differentiate into vascular smooth muscle cells (VSMC) and vascular endothelial cells (VEC), and also secrete vascular endothelial growth factor (VEGF).ConclusionBM-MSCs can migrate to transplanted islet and promote neovascularization. Also, it enhanced allograft immune tolerance of islet grafts via increasing donor lymphocytic chimerism.     

Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice

Transplantation of healthy cells to repair organ damage or replace deficient functions constitutes a major goal of cell therapy. However, the mechanisms by which transplanted cells engraft, proliferate, and function remain unknown. To investigate whether host liver sinusoidal endothelium could be replaced with transplanted liver sinusoidal endothelial cells, we developed an animal model of tissue replacement that utilized a genetic system to identify transplanted cells and induced host-cell perturbations to confer a proliferative advantage to transplanted cells. Under these experimental conditions, transplanted cells engrafted efficiently and proliferated to replace substantial portions of the liver endothelium. Tissue studies demonstrated that transplanted cells became integral to the liver structure and reacquired characteristic endothelial morphology. Characterization of transplanted endothelial cells by membrane markers and studies of cellular function, including synthesis and release of coagulation factor VIII, demonstrated that transplanted cells were functionally intact. Further analysis showed that repopulation of the livers of mice that model hemophilia A with healthy endothelial cells restored plasma factor VIII activity and corrected their bleeding phenotype. Our studies therefore suggest that transplantation of healthy endothelial cells should be considered for cell therapy of relevant disorders and that endothelial reconstitution with transplanted cells may offer an excellent paradigm for defining organ-specific pathophysiological mechanisms.     

Co‐culture of adipose‐derived stem cells and endothelial cells in fibrin induces angiogenesis and vasculogenesis in a chorioallantoic membrane model

Neovascularization of adipose tissue equivalents is a crucial step in successful adipose tissue engineering, since insufficient vascularization results in graft resorption in an in vivo situation. A possible cellular approach to overcome this limitation is the co‐implantation of adipose‐derived stem cells (ASCs) with endothelial cells to stimulate the formation of a vascular network. We investigated the potential of ASCs derived from human abdominal fat tissue co‐cultured with endothelial progenitor cells (EPCs) from human peripheral blood to stimulate neovascularization of fibrin constructs on the chorioallantoic membrane (CAM) of fertilized chicken eggs, in direct comparison to human umbilical vein endothelial cells (HUVECs). After 9 days of incubation, cell–fibrin constructs were explanted and histologically evaluated with respect to ingrowth of avian blood vessels into the construct and formation of human blood vessels by co‐implanted endothelial cells. When administered on the CAM, ASCs successfully guided host vasculature into the construct (angiogenesis) and guided formation of capillary‐like structures by co‐implanted human endothelial cells (vasculogenesis), with HUVECs being superior to EPCs, leading to a perfused avian and human capillary network within the fibrin construct. However, the results also showed that perfused human blood vessels were only observed near the CAM compared to unperfused capillary‐like structures near the top of the construct, indicating that perfusion of the cell–fibrin construct takes longer than 9 days. In conclusion, as blood vessel formation is an essential step during adipogenic differentiation, the data support our hypothesis that cellular communication between transplanted ASCs and endothelial cells is beneficial for vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

异基因外周血造血干细胞移植治疗急性再生障碍性贫血--一例报告附文献复习

目的探索异基因外周血造血干细胞移植治疗急性再生障碍性贫血(再障)的疗效及其长期造血重建.方法1例急性再障患者,30岁,供者为其胞弟,HLA配型完全相合.动员方案G-CSF250?μg/d×6d.预处理方案环磷酰胺(CTX)50mg·kg-1·d-1×4d,抗胸腺细胞球蛋白(ATG)20mg·kg-1·     

Human embryonic stem cells: Possibilities for human cell transplantation

Human embryonic stem (ES) cells serve as a potentially unlimited renewable source for cell transplantation targeted to treat several diseases. One advantage of embryonic stem (ES) cells over other stem cells under research is their apparently indefinite self‐renewal capacity if cultured appropriately, and their ready differentiation into various cell phenotypes of all three germ layers. To date, a number of studies have reported the derivation of specific functional derivatives from human ES cells in vitro. While there have been clinical trials of human embryonal carcinoma (EC) cell‐derived neurons in humans there has been no attempt as yet using human ES cell derivatives. However, the latter have been transplanted into recipient animals. In some cases ES‐derived cells were shown to undergo further maturation, displayed integration with host tissue and even ameliorated the disease condition in the animal model. Recently, it has been reported that human ES cells can be genetically manipulated. Such procedures could be used to direct differentiation to a specific cell type or to reduce graft rejections by the modification of immune responses. This review highlights some of the recent advances in the field and the challenges that lie ahead before clinical trials using ES‐derived cells can be contemplated.     

Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice

The potential of using bone marrow (BM)-derived human stromal cells for ex vivo gene therapy of hemophilia A was evaluated. BM stromal cells were transduced with an intron-based Moloney murine leukemia virus (Mo-MuLV) retroviral vector that contained the B domain-deleted human factor VIII (FVIIIdeltaB) cDNA. This FVIII-retroviral vector was pseudotyped with the gibbon ape leukemia virus envelope (GALV-env) to attain higher transduction efficiencies. Using optimized transduction methods, high in vitro FVIII expression levels of 700 to 2500 mU of FVIII/10(6) cells per 24 hr were achieved without selective enrichment of the transduced BM stromal cells. After xenografting of 1.5-3 x 106 engineered BM stromal cells into the spleen of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice, human plasma FVIII levels rose to 13 +/- 4 ng/ml but declined to basal levels by 3 weeks postinjection because of promoter inactivation. About 10% of these stromal cells engrafted in the spleen and persisted for at least 4 months after transplantation in the absence of myeloablative conditioning. No human BM stromal cells could be detected in other organs. These findings indicate that retroviral vector-mediated gene therapy using engineered BM stromal cells may lead to therapeutic levels of FVIII in vivo and that long-term engraftment of human BM stromal cells was achieved in the absence of myeloablative conditioning and without neo-organs. Hence, BM stromal cells may be useful for gene therapy of hemophilia A, provided prolonged expression can be achieved by using alternative promoters.