Therapeutic potential of embryonic stem cells

Nearly 20 years after murine embryonic stem cells (mESC) were isolated, the first report of the derivation of human embryonic stem cells (hESC) in 1998 spawned the field of hESC research [Evans MJ, Kaufman MH, Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292 (5819): 154-6; Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282 (5391): 1145-7.]. Although this field is only in its infancy, hESC represent a theoretically inexhaustible source of precursor cells that could be differentiated into any cell type to treat degenerative, malignant, or genetic diseases, or injury due to inflammation, infection, and trauma. This pluripotent, endlessly dividing cell has been hailed as a possible means for treating diabetes, Parkinson's disease, Alzheimer's, spinal cord injury, heart failure, and bone marrow failure. But the regenerative medicine applications of embryonic stem cells are only one facet of hESC therapeutic potential. Human ESC are an invaluable research tool to study development, both normal and abnormal, and can serve as a platform to develop and test new therapies. In addition to discussing the therapeutic potential of hESC, this chapter will cover limitations to using hESC for replacement cell therapy, strategies to overcome these limitations, and alternative methods of deriving hESC.     

Cellular mechanical properties reflect the differentiation potential of adipose-derived mesenchymal stem cells

The mechanical properties of adipose-derived stem cell (ASC) clones correlate with their ability to produce tissue-specific metabolites, a finding that has dramatic implications for cell-based regenerative therapies. Autologous ASCs are an attractive cell source due to their immunogenicity and multipotent characteristics. However, for practical applications ASCs must first be purified from other cell types, a critical step which has proven difficult using surface-marker approaches. Alternative enrichment strategies identifying broad categories of tissue-specific cells are necessary for translational applications. One possibility developed in our lab uses single-cell mechanical properties as predictive biomarkers of ASC clonal differentiation capability. Elastic and viscoelastic properties of undifferentiated ASCs were tested via atomic force microscopy and correlated with lineage-specific metabolite production. Cell sorting simulations based on these "mechanical biomarkers" indicated they were predictive of differentiation capability and could be used to enrich for tissue-specific cells, which if implemented could dramatically improve the quality of regenerated tissues.     

肺干细胞研究进展

干细胞研究是现代医学领域研究热点之一.肺部疾病所导致的不同程度呼吸系统病理改变和功能受损,都伴随着肺组织的修复和重塑过程.对于肺部疾病的干细胞研究和应用尚有许多问题有待进一步的明确和探索.肺干细胞包括了肺组织自身的干细胞修复和肺外组织来源的干细胞修复.肺组织内的干细胞包括肺内上皮性干细胞、肺问充质干细胞、肺侧群细胞;其中肺内上皮性干细胞又包括了基底细胞、Clara细胞、Ⅱ型肺泡上皮细胞、"芽孢"样细胞.肺外组织来源的干细胞修复包括骨髓间充质干细胞和造血干细胞.干细胞治疗方法在临床上有巨大的应用前景.     

小肠干细胞与其微环境

人类和啮齿类动物的小肠上皮终生更新不断，这种更新是由位于小肠隐窝底部的多潜能干细胞驱动的。小肠上皮细胞的凋亡脱落及受损坏死与干细胞增殖、分化之间的动态平衡，在维持肠道屏障结构和功能的完整以及损伤后的修复中扮演着重要角色。小肠上皮是单层柱状上皮结构，在人类、小鼠和大鼠，肠上皮在从功能上可分为两个不连续的单位——绒毛分化单位和隐窝增殖单位，干细胞增殖、分化移行路线清晰，使之成为研究成体干细胞体内分化机制的良好模型。定位和分析小肠干细胞的生存环境并阐明细胞分化具体的分子机制，将有助于理解环境因素对干细胞生物学性状的影响。本文就小肠干细胞的分化机制与微环境的关系作一综述。     

How we mobilize haemopoietic stem cells

Mobilization and collection of haemopoietic stem and progenitor cells (HSPC) is the cornerstone of autologous and allogeneic stem cell transplantation for a wide variety of haematological and some non-haematological malignancies. Centres providing this service face the challenge of optimizing the likelihood of successful collection of transplantable doses of cells, while maximizing the efficiency of the apheresis unit and minimizing the risk of toxicity as well as mobilization failure. Recent developments in the understanding of the molecular mechanisms of mobilization have led to the emergence of novel strategies for HSPC mobilization, which may assist in meeting these imperatives. The task for clinicians is how to incorporate the use of these strategies into practice, in the light of emerging evidence for efficacy and safety of these agents. Herein, the literature is reviewed, and a proposed algorithm for HSPC mobilization is presented.     

Efficient replacing of fetal bovine serum with human platelet releasate during propagation and differentiation of human bone-marrow-derived mesenchymal stem cells to functional hepatocyte-like cells

Objectives  The aim of this study was to find out substitution effect of fetal bovine serum (FBS) with human platelet releasate (HPR) as a major growth factor source during expansion and differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) into hepatocytes.
Methods  Propagation and differentiation potential of hBMSCs into hepatocyte-like cells in a medium fortified with HPR instead of FBS were investigated with morphological, cytochemical and molecular experiments.
Results  Multiplex analysis showed that HPR was more efficient than FBS in supporting hBMSC outgrowth. The proliferation rate of MSC in presence of HPR (derived from 10⁹ platelets/ml) was about threefold greater than that of FBS ( P <  0·001). Despite the differences in MTT value, hBMSCs-driven HPR or FBS did not differ in terms of gross morphology, immunophenotype and osteogenic differentiation potential. Hepatic differentiation of hBMSCs was successfully performed in the media enriched with HPR. Immunoreactivity of cells with monoclonal antibodies against for albumin and α-fetoprotein (AFP) was even more positive in hepatocytes differentiated in presence of HPR as compared to that of FBS. The gene expression of albumin, AFP and cytokeratin-18 at mRNA levels in differentiated cells attest to supporting role of HPR in hepatic differentiation media. These findings were further confirmed with greater urea production (approximately twofold) in the culture media of cells differentiated under HPR compared to that in FBS ( P <  0·001).
Conclusion  Human platelet releasate is an efficient and safe substitute for FBS in culture media used for expansion and differentiation of hBMSCs to hepatocyte.     

Double-negative regulatory T cells induce allotolerance when expanded after allogeneic haematopoietic stem cell transplantation

Double-negative (DN) regulatory T cells (Tregs) are specialized T lymphocytes involved in the down-modulation of immune responses, resulting in allotolerance after allogeneic haematopoietic stem cell transplantation (HSCT). Most of the properties of DN Tregs were identified in murine models, including the unique ability to suppress alloreactive syngeneic effector T cells in an antigen-specific manner via Fas/Fas-ligand interactions. We investigated the behaviour of DN Tregs following human allogeneic HSCT with regard to occurrence of graft-versus-host disease (GvHD) and restoration of T-cell receptor repertoire in a cohort of 40 patients. The frequency of DN Tregs and CD4/CD8 TCR repertoire was measured serially and at the time of diagnosis of GvHD by flow cytometry. Analysis demonstrated a positive correlation between degree of alloreactivity, as measured by grade of GvHD, and the number of variable beta chain (Vβ) family expansions in both T-cell populations. We also found that a deficiency of DN Tregs was associated with an increased number of Vβ family expansions, and most importantly, with the occurrence of GvHD. All individuals who demonstrated more than 1% DN Tregs did not develop GvHD, providing evidence that DN Tregs participate in peripheral tolerance to prevent GvHD when expanded after allogeneic HSCT.     

大鼠胰腺导管来源干细胞向胰岛素分泌细胞分化的体外培养

目的:研究在体外条件下从大鼠胰腺导管分离的干细胞向胰岛素分泌细胞分化的产物细胞的形态、表型及功能.方法:采用胶原酶原位消化法消化大鼠胰腺,差异贴壁法培养出胰腺导管来源千细胞(PDSCs),对其进行形态学与表型鉴定.采用无血清培养基,添加Matrigel、exendin-4诱导干细胞向胰岛素分泌细胞分化,鉴定产物细胞的形...     

Basics and applications of stem cells in the pancreas

Enormous efforts have been made to establish pancreatic stem/progenitor cells as a source for regenerative medicine for the treatment of diabetes mellitus. In recent years, it has been recognized that the self-renewal of beta cells is the dominant process involved in postnatal beta-cell regeneration and expansion. Nevertheless, several in-vitro studies have suggested that ductal or as yet unidentified cells are candidates for pancreatic stem/progenitor cells that can differentiate into multilineage cells, including insulin⁺ cells. The question remains as to whether beta cells are generated postnatally from stem/progenitor cells other than pre-existing beta cells. Furthermore, mutated pancreatic stem cells are considered to be prospective candidates for cancer stem cells or tumor-initiating cells. This review highlights recent progress in pancreatic stem/progenitor cell research.     

In utero transplantation of haemopoietic stem cells

In utero haematopoietic stem cell transplantation is a potentially valuable therapeutic approach that strives to take advantage of biological opportunities for cellular transplantation that exist in the early gestational fetus. However, with the exception of severe combined immunodeficiency, clinical application has been limited by minimal or no engraftment, suggesting the presence of significant barriers to engraftment within the fetal environnment. Research directed toward elucidating these barriers is progressing, and there is hope that the barriers to engraftment can be overcome in the near future. In the meantime, there are a limited number of specific disorders that are biologically favourable and may be amenable to treatment by this approach using conventional techniques. In the future, strategies that improve the competitive capacity of donor cells or the use of pre-natal transplantation to induce donor-specific tolerance followed by post-natal non-myeloablative enhancement of donor chimerism may allow broad clinical application of this approach.     

干细胞、肿瘤干细胞与肿瘤发生

肿瘤干细胞是肿瘤研究的一个新热点,指出肿瘤可能是由肿瘤干细胞产生,肿瘤干细胞则由正常干细胞恶变形成.正常干细胞的特有性状,使其较成体细胞更易成为肿瘤发生的靶细胞.干细胞可能经基因突变、异常不对称分裂和细胞融合转化为肿瘤干细胞.利用不同的蛋白标志物或荧光探针,通过流式细胞仪分选是发现肿瘤干细胞的主要方法.已证实的肿瘤干细胞皆具有强大的自我更新和增殖能力,以及细胞分化潜能.针对肿瘤干细胞的检测和杀伤,可能为肿瘤早期诊断和治疗带来希望.     

人肺成纤维母细胞表达骨髓间充质干细胞特性的研究

目的研究原代培养的人肺成纤维母细胞体外分化特性。方法于2006年3月至2006年10月在清华大学第一附属医院中心实验室,将原代培养的人肺成纤维母细胞分别在成骨细胞培养基(含地塞米松,维生素C和β-磷酸甘油)和成脂肪细胞培养基(含马血清,地塞米松和胰岛素)作用下进行分化诱导。组织化学方法行碱性磷酸酶和钙化斑块染色,Westernblotting法测定骨桥蛋白表达,油红染色鉴定脂肪细胞形成。结果在成骨细胞培养基诱导下,细胞内碱性磷酸酶表达增加,于第14天可见大量钙盐沉积和骨桥蛋白表达增加。在成脂肪细胞培养基作用第14天,部分细胞内出现脂肪小滴聚集。结论人肺成纤维母细胞具有多分化潜能,能够在一定条件下向成骨细胞和脂肪细胞分化,表现出骨髓间充质干细胞的特性。     

Cotransplantation of HLA-identical mesenchymal stem cells and hematopoietic stem cells in Chinese patients with hematologic diseases

Mesenchymal stem cells (MSCs) may be employed to support hematopoietic reconstitution and mitigate graft-vs.-host disease (GVHD) in transplantation of hematopoietic stem cells (HSCs). The aim of this study was to explore the feasibility and safety of cotransplantation culture-expanded MSCs and HSCs from the same human leukocyte antigen (HLA)-identical sibling donor in Chinese patients with hematologic diseases. Bone marrow mononuclear cells from healthy donors were cultured and expanded ex vivo. Immunophenotype, adipogenic and osteogenic differentiation potential, and karyotype of the harvested MSCs were detected on those who had been cotransplanted with HSCs and MSCs from the same donor. Hematopoietic reconstitutions, complications, and clinical outcomes were observed after cotransplantation in these patients. (1.77 ± 0.40) × 10⁶/kg (donor’s weight) MSCs were successfully expanded from 23.6 ± 5.96 ml of bone marrow samples. They had normal karyotypes with bi-lineages differentiation potential, and were CD73, CD90, and CD105 positive. Twelve patients underwent cotransplantation with no observable adverse response during and after the infusion of MSCs. Hematopoietic reconstitutions were rapid. Two patients developed grade II–IV acute GVHD, and two extensive chronic GVHD. Four patients suffered from cytomegalovirus infection but were cured eventually. Up to now, seven patients have been followed as long as 29–57 months and five patients died. It is concluded that MSCs can be expanded effectively by culture and it is safe and feasible to cotransplant patients with allogenic culture-expanded MSCs and HSCs.     

Mesenchymal stem cells and the lung

Mesenchymal stem cells (MSC) are a population of tissue‐resident adult progenitor cells that were originally identified in bone marrow, but have now been identified in many organs including the lung. Although their precise role in organ function remains incompletely defined, mounting evidence suggests that they are an important component of the parenchymal progenitor cell niche and orchestrate organ homeostasis and repair following injury. In this review, what is known about MSC biology will be outlined with particular emphasis on lung biology, and the therapeutic potential of MSC‐based cell therapy will also be highlighted.     

In vivo haematopoietic potential of human neural stem cells

The fetal sheep model was used to compare the in vivo haematopoietic potential of human neural stem cells (NSC) versus bone marrow (BM)-derived haematopoietic stem cells (HSC). To this end, sheep were transplanted with either 8 x 10(5) NSC (n = 11) or HSC, CD34(+)Lin(-) (n = 5), and subsequently analysed for haematopoietic chimaerism. While HSC-transplanted sheep displayed robust donor-derived haematopoiesis starting at less than 2 months post-transplant, NSC recipients exhibited haematopoietic engraftment at much later time points. Nevertheless, chimaerism persisted in both groups throughout the course of this study. Transplantation of secondary recipients with human CD45(+)/HLA-DR(+) cells from the BM of NSC primary recipients at 14 and 16 months post-transplant demonstrated that long-term engrafting HSC were present in these animals. At 6 months post-transplant, both NSC- and HSC-transplanted sheep were mobilised with granulocyte colony-stimulating factor. In contrast to HSC-transplanted animals, levels of human blood cells in peripheral blood of NSC-transplanted sheep remained low throughout mobilisation. Our results show that, although human NSC were able to give rise to multilineage haematopoiesis in our model, the levels, timing of blood cell production and the ability to respond to cytokine mobilisation were different, suggesting that human NSCs latent haematopoietic potential is inherently different from that of true HSC.     

Therapeutic potential of mesenchymal stem cells overexpressing human forkhead box A2 gene in the regeneration of damaged liver tissues

Background and Aim: Although a liver transplantation is considered to be the only effective long‐term treatment in many cases of liver diseases, it is limited by a lack of donor organs and immune rejection. As an autologous stem cell approach, this study was conducted to assess whether forkhead box A2 (Foxa2) gene overexpression in bone marrow‐derived mesenchymal stem cells (MSC) could protect the liver from hepatic diseases by stimulating tissue regeneration after cell transplantation. Methods: Rat MSC (rMSC) were isolated, characterized, and induced to hepatocytes that expressed liver‐specific markers. Four different treatments (control [phosphate‐buffered saline], rMSC alone, rMSC/pIRES–enhanced green fluorescent protein (EGFP) vector, and rMSC/pIRES–EGFP/human Foxa2) were injected into the spleen of carbon tetrachloride‐injured rats. Biochemical and histological analyses on days 30, 60, and 90 post‐transplantation were performed to evaluate the therapeutic capacities of MSC overexpressing hFoxa2. Results: rMSC transfected with hFoxa2 were induced into hepatogenic linage and expressed several liver‐specific genes, such as, Foxa2, α‐fetoprotein, cytokeratin‐18, hepatocyte nuclear factor‐1α, and hepatocyte growth factor. A group of animals treated with MSC/hFoxa2 showed significant recovery of liver‐specific enzyme expressions to normal levels at the end of the study (90 days). Furthermore, when compared to the fibrotic areas of the samples treated with MSC alone or MSC/vector, the fibrotic area of the samples treated with rMSC/hFoxa2 for 90 days significantly decreased, until they were completely gone. Conclusions: Human Foxa2 efficiently promoted the incorporation of MSC into liver grafts, suggesting that hFoxa2 genes could be used for the structural or functional recovery of damaged liver cells.     

脐血干细胞治疗缺血性卒中

缺血性卒中是目前全球致死率和致残率最高的主要疾病之一.传统治疗方法(如溶栓治疗)存在时间窗窄、疗效不佳等缺陷.脐血干细胞移植作为一种细胞治疗,为难治性神经系统疾病的治疗带来希望.     

脐血干细胞治疗缺血性卒中

缺血性卒中是目前全球致死率和致残率最高的主要疾病之一.传统治疗方法(如溶栓治疗)存在时间窗窄、疗效不佳等缺陷.脐血干细胞移植作为一种细胞治疗,为难治性神经系统疾病的治疗带来希望.