Ex vivo manipulation of hematopoietic stem and progenitor cells

Approaches to manipulate peripheral blood progenitor cells (PBPC) ex vivo currently include the selection of CD34+ cells as a means to purge contaminating tumor cells from leukapheresis preparations or to provide a homogeneous starting population for the expansion of hematopoietic progenitor cells as well as the induction of postprogenitor cells of either the myeloid or megakaryocytic lineage. The latter cell populations might be used for an additional transplantation together with PBPC to possibly shorten the period of aplasia. In addition, ex vivo expansion of CD34+ cells can be used to generate autologous tumor-antigen-presenting dendritic cells for immunotherapeutic approaches aiming to treat minimal residual disease following high-dose chemotherapy.     

Recombinant HoxB4 fusion proteins enhance hematopoietic differentiation of human embryonic stem cells

Enforced expression of the HoxB4 gene promotes expansion of hematopoietic stem cells (HSCs) and enhances hematopoietic development of both murine and human embryonic stem (ES) cells. HoxB4- expanded HSCs have also been shown to retain their normal potential for differentiation and longterm self-renewal in vivo without the development of leukemia, suggesting that manipulation of HoxB4 expression might represent an effective way to expand functional HSCs for use in transplantation medicine. However, the genetic modification of cells poses clinical concerns, including a potentially increased risk of tumor genicity. Constitutive high-level ectopic viral expression of HoxB4 can also produce perturbations in the lineage differentiation of HSCs, an indication that uncontrolled HoxB4 manipulation may not be a satisfactory therapeutic strategy. Here we demonstrate that recombinant HoxB4 protein fused with a triple protein transduction domain (tPTD) promotes hematopoietic development of hES cells. The tPTD-HoxB4 protein enhanced the development of erythroid, myeloid, and multipotential progenitors in both early- and late-stage embryoid bodies (EBs). This effect varied considerably between different hES cell lines. Addition of the tPTD-HoxB4 protein did not alter the globin gene expression pattern; progeny derived from hES cells expressed high levels of embryonic (epsilon) and fetal (gamma) globin genes with or without tPTD-HoxB4 treatment. CD34+ cells derived from hES cells engrafted in bone marrow when transplanted into fetal CD1 mice, although supplementation of the differentiation medium with tPTD-HoxB4 protein did not result in increased repopulating capacity. This suggests that other gene(s), together with HoxB4, are required for generating more competitive HSCs. In summary, our study demonstrates that the tPTD-HoxB4 protein can be used with other recombinant proteins to efficiently generate transplantable HSCs from human ES cells.     

Ex vivo conditions for self-replication of human hepatic stem cells

Human hepatic stem cells (hHpSCs), identifiable by a unique antigenic profile, have been isolated from human livers and established ex vivo under expansion conditions permissive for self-replication. The conditions consist of a substratum of type III collagen, ideally on Transwell inserts, and Kubota's medium, a serum-free medium developed for hepatic progenitors. Under these conditions the cells demonstrated a doubling time of approximately 24 h, generating at least a 16-fold increase in cell number within 7-10 days; were stable at confluence for up to 2 weeks; could be passaged, if on type III collagen, to initiate colonies that went through log-phase growth and saturation density kinetics; and expressed telomerase, indicative of regenerative capacity. The hHpSC colonies remained morphologically and phenotypically stable throughout expressing epithelial cell adhesion molecule, neural cell adhesion molecule, albumin, cytokeratins 8, 18, and 19, but not alpha-fetoprotein, or intercellular adhesion molecule-1 (ICAM-1). Those maintained under self-replication conditions for more than a month were transplanted and found to engraft in the livers of SCID/nod mice yielding human liver tissue expressing adult liver-specific proteins. The conditions for self-replication should offer ideal culture conditions for generating large numbers of hHpSCs for use in commercial and clinical programs.     

人主动脉-性腺-中肾区基质细胞诱导胚胎干细胞分化为造血干细胞及体内造血重建

背景：研究证实多种造血生长因子、基质细胞饲养层及其条件培养液可促进胚胎干细胞向造血干细胞分化。 目的：以人主动脉-性腺-中肾(aorta-gonad-mesonephros,AGM)区基质细胞为饲养层体外诱导小鼠胚胎干细胞分化为造血干细胞,并比较不同移植途径对造血干细胞体内造血重建能力的影响。 方法：将小鼠E14 胚胎干细胞诱导为拟胚体,采用Transwell非接触共培养体系在人AGM区基质细胞饲养层上诱导6 d,接种NOD-SCID小鼠检测体内致瘤性。再将诱导后的拟胚体细胞移植经致死量60Co γ射线辐照的BALB/C雌鼠,受鼠随机分为静脉移植组、骨髓腔移植组、照射对照组及正常对照组。 结果与结论：拟胚体细胞经人AGM区基质细胞诱导后Sca-1⁺c-Kit⁺细胞占(13.12±1.30)%。NOD-SCID小鼠皮下接种经人AGM区基质细胞诱导的拟胚体细胞可出现畸胎瘤,经骨髓腔接种未见肿瘤形成。静脉移植组动物全部死亡,骨髓腔移植组生存率为55.6%,移植后21 d外周血象基本恢复,存活受鼠检测到供体来源Sry基因。提示小鼠胚胎干细胞经人AGM区基质细胞诱导分化的造血干细胞通过骨髓腔移植安全并具有一定的造血重建能力。     

Ex vivo expansion of a hematopoietic stem cell on a murine stromal cell by 3D micro-pillar device

Stromal cells alter their mode of attachment, cellular shape, and protein expression when placed on square arrays of micro-pillars. All the pillars we studied had 50 μm diameters, 85 μm pillar heights, were separated by 50 μm, and had an identical surface chemistry. We found that these micro-pillars provided many opportunities for mechanical interlocking and were more suitable attachment matrixes for cell adhesion and stretching of the overlying biomaterials. When the feeder layer cells of hematopoietic stem cells (HSCs) were cultured into the micro-pillar device, they could screen more hematopoietic cytokines, such as interleukin-3 (IL-3), and laminin into the medium. Consequently, the micro-pillar device provides a greater degree of HSCs expansion relative to the 25 T flask. The maximal expansion of the HSCs and the colony-forming unit (CFU) on the micro-pillar device increased 62.72-fold and 16.95-fold for 28 day culture, but there were only 58.08-fold and 6.8-fold expansion on the 25 T flask. The results showed a significantly higher expansion in the pillar device compared to the 25 T flask; moreover, the stemness was maintained. Therefore, the 3D micro-pillar device appears to be a more suitable culture substrate for HSCs expansion ex vivo.     

成人骨髓间充质干细胞对造血干细胞体外增殖的影响

目的研究骨髓间充质干细胞（MSC）对脐带血（CB）CD34^＋细胞体外增殖和造血重建能力的影响。方法取人骨髓单个核细胞贴壁培养．梭形细胞完全融合后传代，用流式细胞仪检测免疫表型；将CBCD34^＋细胞接种到MSC或其他培养液中．比较不同培养条件对造血干细胞扩增能力、集落形成能力及黏附分子表达的影响。结果在加入IL-3的培养体系中．在MSC和细胞因子作用下，CD34^＋细胞扩增7d和14d后，有核细胞（NC）、CD34^＋细胞和CDl33^＋细胞数，实验组均显著多于对照组。CD34＋细胞在未加入IL-3的培养体系中培养8d后，实验组NC、CD34^＋细胞、CD34^＋CD38-细胞和造血祖细胞集落扩增倍数均显著高于对照组。扩增后CD34^＋细胞的ALCAM、VLA-α4、VLA-α5、VLA-β1、HCAM、PECAM和LFA-1表达较扩增前无显著变化。结论MSC可为造血干细胞（HSC）体外扩增提供适宜的微环境，有助于CD34^＋细胞体外增殖并抑制HSC分化，保持其造血重建潜能和归巢能力。     

造血干/祖细胞体外扩增的最新研究进展

造血干/祖细胞体外扩增方法的快速发展为造血干/祖细胞广泛应用于临床开辟了广阔的前景,就造血干/祖细胞体外扩增的方法和培养系统的最新进展做一综述。     

造血干/祖细胞体外扩增的最新研究进展

造血干/祖细胞体外扩增方法的快速发展为造血于/祖细胞广泛应用于临床开辟了广阔的前景,就造血干/祖细胞体外扩增的方法和培养系统的最新进展做一综述.     

Enhance tumor radiosensitivity by intracellular delivery of eukaryotic translation initiation factor 4E binding proteins

PTEN (phosphatase and tensin homologue deleted on chromosome ten)/PI3K (phosphatidylinositol 3-kinase)/Akt/mTOR (mammalian target of rapamycin) signaling pathway, which is commonly dysregulated in a broad array of human malignancies, controls the assembly of eukaryotic translation initiation factor 4F (eIF4F) complex through regulation of eIF4E binding proteins (4E-BPs) phosphorylation. And accumulated data over the past two decades implicated eIF4F complex as one of the promising targets for anticancer therapy. It has been confirmed that the translation initiation of mRNA coding for hypoxia-inducible factor-1α (HIF-1α) and survivin, which had been considered as the two major determinants of tumor radiosensitivity, are both controlled by eIF4F complex. Also, eIF4F complex controls the expression of VEGF and bFGF, the two well-known pro-angiogenic factors involved in developing radioresistance. Therefore eIF4F complex plays a pivotal role in regulation of radiosensitivity. In this article, we postulate that cell-permeable, phosphorylation-defective 4E-BP fusion proteins, which could be prepared by substituting the mTOR recognition motif located in N-terminal of 4E-BPs with protein transduction domain from HIV-1 TAT, HSV-1 VP22 or PTD4, could not only inhibit tumor growth but also enhance tumor response to radiation therapy through disruption of eIF4F complex assembly. In our opinion, the recombinant fusion proteins are superior to mTOR inhibitors for they do not cause immunosuppression, do not lead to Akt activation, and could be easily prepared by prokaryotic expression. If the hypothesis was proved to be practical, the cell-permeable, phosphorylation-defective 4E-BP fusion proteins would be widely used in clinical settings to improve tumor response to radiotherapy in the near future.     

HOXA4 induces expansion of hematopoietic stem cells in vitro and confers enhancement of pro-B-cells in vivo

Members of the homeobox (Hox) gene family are known to mediate expansion of hematopoietic stem cells (HSCs) and progenitors. The absence of oncogenic properties promoted HOXB4 as prime candidate in the quest to expand HSCs for clinical purposes. Despite its potential to expand HSCs, studies with mutant mice showed that Hoxb4 is not essential for HSC generation and function under physiological conditions. Expression studies and the existence of functional redundancy in particular between paralog Hox genes suggest that HOXA4 might have potent properties to expand HSCs. Here we measured the ability of HOXA4 to promote ex vivo expansion of HSCs and progenitors using retrovirus-mediated overexpression. Our results provide evidence that HOXA4-transduced HSCs and primitive progenitors expand in culture conditions and demonstrate that the potential of expanded HOXA4 HSCs to give rise to mature myeloid and lymphoid progeny in normal proportions remained intact. Interestingly, constitutive overexpression of HOXA4 resulted in an unbalanced expansion of lymphoid/myeloid progenitors in bone marrow chimeras favorable to B-cell progenitors responsive to interleukin-7. This expansion was specific for these progenitors and not for the more primitive Whitlock-Witte-initiating cells. These data indicate that early stages of B-cell development associated with proliferation are in particular sensitive to HOXA4. Thus, this study supports the potential use of HOXA4 to expand both HSCs and B-cell progenitor populations for therapeutic strategies.     

Ex vivo expansion and prophylactic infusion of CMV-pp65 peptide-specific cytotoxic T-lymphocytes following allogeneic hematopoietic stem cell transplantation.

Cytomegalovirus reactivation and infection post-allogeneic hematopoietic stem cell transplant continue to cause morbidity and mortality. Current pharmacologic therapies are limited by side effects. Adoptive transfer of ex vivo generated cytomegalovirus-specific T cells has the potential to restore immunity, prevent cytomegalovirus, and circumvent the need for pharmacologic therapies. We have generated donor-derived cytomegalovirus-specific cytotoxic T cells using dendritic cells pulsed with the HLA-A2 restricted nonapeptide NLVPMVATV (NLV) derived from the cytomegalovirus-pp65 protein. These cytotoxic T cells have been given prophylactically to 9 recipients aged 4 to 65 years on or after day 28 post-allogeneic hematopoietic stem cell transplant. Only 2 of 9 recipients received T cell depletion in vivo or in vitro. There were no immediate adverse reactions to the infusions. During 97-798 days of follow-up, 2 recipients developed cytomegalovirus reactivation; neither developed cytomegalovirus disease or required pharmacotherapy. Three recipients developed acute graft versus host disease after infusion. Two recipients died, 1 from thrombotic thrombocytopenia purpura secondary to cyclosporine, 1 from complications of graft versus host disease. A transient increase in numbers of cytomegalovirus-specific T cells demonstrated by NLV-tetramer binding was seen in 6 recipients. Prophylactic adoptive transfer of NLV-specific T cells is safe and may be effective in preventing cytomegalovirus reactivation.     

人AGM区及胎肝基质细胞促进小鼠胚胎干细胞向造血干细胞分化和体内造血重建

目的: 探讨小鼠胚胎干细胞(ESCs)经人主动脉-性腺-中肾(AGM)区及胎肝(FL)基质细胞程序诱导后,向造血干细胞(HSCs)分化的效率及其造血功能。方法: 将E14 ESCs诱导为拟胚体(EB),并在人AGM区及FL基质细胞饲养层上进一步诱导分化,培养6 d后收集细胞检测Sca-1⁺c-Kit⁺细胞含量、分析造血细胞集落形成能力及致瘤性。再将不同诱导阶段的EB来源细胞移植经致死量 γ射线辐照的BALB/c雌鼠,观察生存率、植入状况和造血重建。结果: (1)EB来源细胞经人AGM区及FL基质细胞程序诱导后Sca-1⁺c-Kit⁺细胞含量为(21.96±2.54)%,造血集落总数为(520±52)/10⁵cells,明显优于诱导前及人AGM区基质细胞初步诱导者(P＜0.05)。(2)NOD-SCID小鼠接种经人AGM区及FL基质细胞诱导的ESCs未见畸胎瘤。(3)BALB/c雌鼠移植经人AGM区及FL基质细胞诱导的EB来源细胞后生存率77.8%,14 d外周血细胞计数明显改善,存活受鼠均检测到供体来源sry基因,而移植人AGM区基质细胞诱导的EB细胞者15 d内全部死亡。结论: 人AGM区及FL基质细胞能促进小鼠ESCs定向分化为HSCs,有效重建体内造血功能。     

Ex vivo culture of human cord blood hematopoietic stem/progenitor cells adversely influences their distribution to other bone marrow compartments after intra-bone marrow transplantation

Intra-bone marrow injection is a novel strategy for hematopoietic stem cell transplantation. Here, we investigated whether ex vivo culture of cord blood hematopoietic stem/progenitor cells influences their reconstitution in bone marrow after intra-bone marrow transplantation. Freshly isolated AC133(+) cells or cells derived from AC133(+) cells cultured with cytokines (stem cell factor, flt-3 ligand, and thrombopoietin) for 5 days were injected into the bone marrow of the left tibia in irradiated NOD/SCID mice. In the bone marrow of the injected left tibia, the engraftment levels of human CD45(+) cells at 6 weeks after transplantation did not differ considerably between transplantation of noncultured and cytokine-cultured cells. However, the migration and distribution of transplanted cells to the bone marrow of other, noninjected bones were extremely reduced for cytokine-treated cells compared with noncultured cells. Similar findings were observed for engraftment of CD34(+) cells. Administration of granulocyte colony-stimulating factor to mice after transplantation induced the migration of cytokine-cultured cells to the bone marrow of previously aspirated bone but not to other intact bones. These data suggest that ex vivo manipulation of hematopoietic progenitor/stem cells significantly affects their migration properties to other bone marrow compartments after intra-bone marrow transplantation. Our data raise a caution for future clinical applications of the intra-bone marrow transplantation method using ex vivo-manipulated hematopoietic stem cells.     

人正常骨髓CD34~ 造血细胞体外扩增形成粒单系造血祖细胞的能力

CD34 造血细胞以其独特的生物学功能正成为造血调控、造血于细胞移植和基因治疗最理想的靶细胞。本文探讨人正常骨髓CD34 造血细胞在体外扩增形成单个核细胞（MNC）及粒单系集落形成单位（CFU－GM）的能力，采用CIMS－100新型免疫磁性无菌分离术可获得纯度＞90％的CD34 造血细胞，其直接形成CFLJ－GM的能力要较骨髓MNC提高的倍，并且在含EGIIS组合造血生长因子的无基质液培养系统中，CD34 造血细胞在四周内可持续扩增产生大量MNC及CFU－GM，最高分别可达1770倍和48倍，但不同个体问CD34 造血细胞的这种能力差别较大。上述结果提示，CD34 造血细胞在最佳组合HGFs的无基质液培养条件下，能扩增产生大量成熟及晚期造血祖细胞，可以适用于临床治疗的需求，