间充质干细胞过滤分离器制备人羊膜间充质干细胞的研究

自然存在的间充质干细胞数量少,限制了其研究应用。依靠自主发明的间充质干细胞过滤分离器,分离制备了人羊膜间充质干细胞,并对制备的干细胞进行了三维培养扩增。结果表明,制备的干细胞形态长势良好,并能诱导分化为类胰岛样组织。与常规方法相比,干细胞收获率提高了8倍以上,且细胞活性状态良好。间充质干细胞过滤分离器可以批量制备高质量的各种间充质干细胞,有利于高效率地建设各种间充质干细胞库,以促进间充质干细胞的研究应用。     

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: Towards the definition of a mesenchymal stem cell proteomic signature

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2‐DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo‐natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.     

Fat pad-derived mesenchymal stem cells as a potential source for cell-based adipose tissue repair strategies

Background: Mesenchymal stem cells are able to undergo adipogenic differentiation and present a possible alternative cell source for regeneration and replacement of adipose tissue. The human infrapatellar fat pad is a promising source of mesenchymal stem cells with many source advantages over from bone marrow. It is important to determine whether a potential mesenchymal stem‐cell exhibits tri‐lineage differentiation potential and is able to maintain its proliferation potential and cell‐surface characterization on expansion in tissue culture. We have previously shown that mesenchymal stem cells derived from the fat pad can undergo chondrogenic and osteogenic differentiation, and we characterized these cells at early passage. In the study described here, proliferation potential and characterization of fat pad‐derived mesenchymal stem cells were assessed at higher passages, and cells were allowed to undergo adipogenic differentiation. Materials and methods: Infrapatellar fat pad tissue was obtained from six patients undergoing total knee replacement. Cells isolated were expanded to passage 18 and proliferation rates were measured. Passage 10 and 18 cells were characterized for cell‐surface epitopes using a range of markers. Passage 2 cells were allowed to undergo differentiation in adipogenic medium. Results: The cells maintained their population doubling rates up to passage 18. Cells at passage 10 and passage 18 had cell‐surface epitope expression similar to other mesenchymal stem cells previously described. By staining it was revealed that they highly expressed CD13, CD29, CD44, CD90 and CD105, and did not express CD34 or CD56, they were also negative for LNGFR and STRO1. 3G5 positive cells were noted in cells from both passages. These fat pad‐derived cells had adipogenic differentiation when assessed using gene expression for peroxisome proliferator‐activated receptor γ2 and lipoprotein lipase, and oil red O staining. Discussion: These results indicate that the cells maintained their proliferation rate, and continued expressing mesenchymal stem‐cell markers and pericyte marker 3G5 at late passages. These results also show that the cells were capable of adipogenic differentiation and thus could be a promising source for regeneration and replacement of adipose tissue in reconstructive surgery.     

Human amnion-derived mesenchymal stem cells are a potential source for uterine stem cell therapy

Abstract. Objectives: Human amnion is an easy‐to‐obtain novel source of human mesenchymal stem cells, which poses little or no ethical dilemmas. We have previously shown that human amnion‐derived mesenchymal (HAM) cells exhibit certain mesenchymal stem cell‐like characteristics with respect to expression of stem cell markers and differentiation potentials. Materials and methods: In this study, we further characterized HAM cells’ potential for in vivo therapeutic application. Results: Flow cytometric analyses of HAM cells show that they express several stem cell‐related cell surface markers, including CD90, CD105, CD59, CD49d, CD44 and HLA‐ABC, but not CD45, CD34, CD31, CD106 or HLA‐DR. HAM cells at the 10th passage showed normal karyotype. More interestingly, the AbdB‐like HOXA genes HOXA9, HOXA10 and HOXA11 that are expressed in the mesenchyme of the developing female reproductive tract and pregnant uteri are also expressed in HAM cells, suggesting similarities between these two mesenchymal cell types. Progesterone receptor is also highly expressed in HAM cells and expression of genes or proteins in HAM cells could be manipulated with the aid of lentivirus technology or cell‐permeable peptides. To test potentials of HAM cells for in vivo application, we introduced enhanced green fluorescence protein (EGFP)‐expressing HAM cells to mice by intrauterine infusion (into uteri) or by intravenous injection (into the circulation). Presence of EGFP‐expressing cells within the uterine mesenchyme after intrauterine infusion or in lungs after intravenous injection was noted within 1–4 weeks. Conclusions: Collectively, these results suggest that HAM cells are a potential source of mesenchymal stem cells with therapeutic potential.     

Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.     

Proteomic analysis of hypoxia and non-hypoxia secretome mesenchymal stem-like cells from human breastmilk

IntroductionBreastmilk contains proteins and cells which have stem cell properties. The human breastmilk stem cell mimick mesenchymal stem cells and expresses pluripotency genes. The protein level of breastmilk is high in colostrum and gradually subsides in the first year of lactation. The mesenchymal stem cells from breastmilk can be an alternative source of stem cells that can potentially affect cardiovascular therapy. This study aimed to identify the proteomic analysis of secretome mesenchymal stem-like cells under hypoxia compared to non-hypoxia from human breastmilk stem cells.Material and methodsThe human breastmilk was collected from six healthy breastfeeding women and transported to the laboratory under aseptic conditions. The breastmilk cells were isolated then cultured. After 72 h, the human breastmilk stem cells reached confluence then cleaned up and isolated in serum-free media (spheroid) to allow serial passaging every 48 h. The acquisition stem cell was made with flow cytometry. The cells were divided into hBSC secretomes under hypoxia (A) and non-hypoxia (B) and analyzed for LC-MS to identify the peptide structure.ResultsThe human breastmilk cells contained several mesenchymal stem-like cells in density 2.4 × 10⁶ cell/mL for hypoxia and 2 × 10⁶ cell/mL for non-hypoxia conditions. The human breastmilk stem cell surface markers derived from the third cell passage process were 93.77% for CD44, 98.69% for CD73, 88.45% for CD90, and 96.30% for CD105. The protein level of secretome mesenchymal stem -like cells under hypoxia was measured at 5.56 μg/mL and 4.28 μg/mL for non-hypoxia. The liquid chromatography-mass spectrometry analysis identified 130 and 59 peptides from hypoxia and non-hypoxia of the human breastmilk stem cell secretome sequentially. Some important proteomics structures were found in the hypoxic human breastmilk stem cell secretome, such as transforming growth factor-β, VE-cadherin, and caspase.ConclusionThe human breastmilk cells contain mesenchymal stem-like cells and a high concentration of CD44, CD73, CD90, and CD105 as surface markers at third passage culture. The hypoxic hBSC secretome produces a higher protein level compare to non-hypoxia. The transforming growth factor -β was found in the hypoxic hBSC secretome as a modulator of VEGF-mediated angiogenesis.     

人脐带间充质干细胞在急性肾小管坏死模型犬的体内分布及趋化性

目的 观察人脐带间充质干细胞在家犬急性肾小管坏死模型的体内分布及归巢.方法 健康家犬18 只随机分为3 组.模型1 组:肌注新鲜配制的0.2﹪二氯化汞溶液7 ml/kg建立急性肾小管坏死模型,采用经外周静脉注射法输注体外分离培养并用4',6- 二脒基-2- 苯基吲哚(DAPI)标记的人脐带间充质干细胞.模型2 组:造模...     

人羊膜间充质干细胞对四氯化碳诱导小鼠肝损伤定位修复的研究

目的：观察活体染料羧基荧光素乙酰乙酸（CFSE）标记的人羊膜间充质干细胞对四氯化碳诱导小鼠肝损伤模型的定位修复情况。方法：采用胰蛋白酶-胶原酶消化法从羊膜组织中分离间充质干细胞,通过流式细胞术和免疫荧光等方法进行鉴定。模型组按浓度为20μl/g剂量的四氯化碳和橄榄油混合液诱导小鼠肝损伤,治疗组经小鼠尾静脉注射羧基荧光素乙酰乙酸标记的人羊膜间充质干细胞约1×10⁶个/ml。分别取模型组和细胞移植的治疗组小鼠眼球血和肝组织进行相关检测。结果：分离得到纯度较高的羊膜间充质干细胞;冰冻切片免疫荧光显示移植1周后细胞向小鼠受损肝组织定植,CFSE标记的人羊膜间充质干细胞呈绿色荧光;细胞移植后4周,与模型组比较,细胞移植组小鼠血清中天冬氨酸转移酶、丙氨酸氨基转移酶显著降低,而白蛋白明显升高（P< 0.01）;肝组织病理切片模型组小鼠细胞水肿,坏死灶多见,脂肪变性,可见不同程度的炎性细胞浸润;治疗组小鼠肝组织病理学改变和损伤程度有较明显改善;小鼠肝组织冰冻切片的免疫荧光显示移植4周后人羊膜间充质干细胞周围分泌血清白蛋白。结论：羧基荧光素乙酰乙酸标记的人羊膜间充质干细胞可有效改善肝组织的生理功能,为细胞定位移植治疗肝脏疾病的修复情况提供实验数据。     

Angiogenic and Restorative Abilities of Human Mesenchymal Stem Cells Were Reduced Following Treatment With Serum From Diabetes Mellitus Type 2 Patients

This experiment investigated the impact of serum from patients with type 2 diabetes mellitus on the angiogenic behavior of human mesenchymal stem cells in vitro. Changes in the level of Ang‐1, Ang‐2, cell migration, and trans‐differentiation into pericytes and endothelial lineage were monitored after 7 days. The interaction of mesenchymal stem cells with endothelial cells were evaluated using surface plasmon resonance technique. Paracrine restorative effect of diabetic stem cells was tested on pancreatic β cells. Compared to data from FBS and normal serum, diabetic serum reduced the stem cell survival and chemotaxis toward VEGF and SDF‐1α (P < 0.05). Diabetic condition were found to decline cell migration rate and the activity of MMP‐2 and ‐9 (P < 0.05). The down‐regulation of VEGFR‐2 and CXCR‐4 was observed with an increase in the level of miR‐1‐3p and miR‐15b‐5p at the same time. The paracrine angiogenic potential of diabetic stem cells was disturbed via the changes in the dynamic of Ang‐1, Ang‐2, and VEGF. Surface plasmon resonance analysis showed that diabetes could induce an aberrant increase in the interaction of stem cells with endothelial cells. After treatment with diabetic serum, the expression of VE‐cadherin and NG2 and ability for uptake of Dil‐Ac‐LDL were reduced (P < 0.01). Conditioned media prepared from diabetic stem cells were unable to decrease fatty acid accumulation in β‐cells (P < 0.05). The level of insulin secreted by β‐cells was not affected after exposure to supernatant from diabetic or non‐diabetic mesenchymal stem cells. Data suggest diabetes could decrease angiogenic and restorative effect of stem cells in vitro. J. Cell. Biochem. 119: 524–535, 2018. © 2017 Wiley Periodicals, Inc.     

Stromal stem cells from adipose tissue and bone marrow of age‐matched female donors display distinct immunophenotypic profiles

Adipose tissue is composed of lipid‐filled mature adipocytes and a heterogeneous stromal vascular fraction (SVF) population of cells. Similarly, the bone marrow (BM) is composed of multiple cell types including adipocytes, hematopoietic, osteoprogenitor, and stromal cells necessary to support hematopoiesis. Both adipose and BM contain a population of mesenchymal stromal/stem cells with the potential to differentiate into multiple lineages, including adipogenic, chondrogenic, and osteogenic cells, depending on the culture conditions. In this study we have shown that human adipose‐derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) populations display a common expression profile for many surface antigens, including CD29, CD49c, CD147, CD166, and HLA‐abc. Nevertheless, significant differences were noted in the expression of CD34 and its related protein, PODXL, CD36, CD 49f, CD106, and CD146. Furthermore, ASCs displayed more pronounced adipogenic differentiation capability relative to BMSC based on Oil Red staining (7‐fold vs. 2.85‐fold induction). In contrast, no difference between the stem cell types was detected for osteogenic differentiation based on Alizarin Red staining. Analysis by RT‐PCR demonstrated that both the ASC and BMSC differentiated adipocytes and osteoblast displayed a significant upregulation of lineage‐specific mRNAs relative to the undifferentiated cell populations; no significant differences in fold mRNA induction was noted between ASCs and BMSCs. In conclusion, these results demonstrate human ASCs and BMSCs display distinct immunophenotypes based on surface positivity and expression intensity as well as differences in adipogenic differentiation. The findings support the use of both human ASCs and BMSCs for clinical regenerative medicine. J. Cell. Physiol. 226: 843–851, 2011. © 2010 Wiley‐Liss, Inc.     

Platelet Lysates Produced from Expired Platelet Concentrates Support Growth and Osteogenic Differentiation of Mesenchymal Stem Cells

Background

Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells.

Methodology/Principal Findings

In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed.

Conclusion/Significance

Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.     

Amniotic mesenchymal stem cells display neurovascular tropism and aid in the recovery of injured peripheral nerves

Recently, we reported that human amniotic membrane‐derived mesenchymal stem cells (AMMs) possess great angiogenic potential. In this study, we determined whether local injection of AMMs ameliorates peripheral neuropathy. AMMs were transplanted into injured sciatic nerves. AMM injection promoted significant recovery of motor nerve conduction velocity and voltage amplitude compared to human adipose‐derived mesenchymal stem cells. AMM implantation also augmented blood perfusion and increased intraneural vascularity. Whole‐mount fluorescent imaging analysis demonstrated that AMMs exhibited higher engraftment and endothelial incorporation abilities in the sciatic nerve. In addition, the higher expression of pro‐angiogenic factors was detected in AMMs injected into the peripheral nerve. Therefore, these data provide novel therapeutic and mechanistic insights into stem cell biology, and AMM transplantation may represent an alternative therapeutic option for treating peripheral neuropathy.     

Regulation of stemness and stem cell niche of mesenchymal stem cells: Implications in tumorigenesis and metastasis

Human mesenchymal stem cells (MSCs) derived from adult tissues have been considered a candidate cell type for cell‐based tissue engineering and regenerative medicine. These multipotent cells have the ability to differentiate along several mesenchymal lineages and possibly along non‐mesenchymal lineages. MSCs possess considerable immunosuppressive properties that can influence the surrounding tissue positively during regeneration, but perhaps negatively towards the pathogenesis of cancer and metastasis. The balance between the naïve stem state and differentiation is highly dependent on the stem cell niche. Identification of stem cell niche components has helped to elucidate the mechanisms of stem cell maintenance and differentiation. Ultimately, the fate of stem cells is dictated by their microenvironment. In this review, we describe the identification and characterization of bone marrow‐derived MSCs, the properties of the bone marrow stem cell niche, and the possibility and likelihood of MSC involvement in cancer progression and metastasis. J. Cell. Physiol. 222: 268–277, 2010. © 2009 Wiley‐Liss, Inc.     

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte‐like cells in injured livers of SCID mice

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte‐like phenotypes. When transplanted intrasplentically into carbon tetrachloride‐injured livers of SCID mice, EGFP‐tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three‐dimensional architecture, and differentiate into hepatocyte‐like cells expressing human albumin and α‐1‐anti‐trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury. J. Cell. Biochem. 108: 693–704, 2009. © 2009 Wiley‐Liss, Inc.     

人脐带间充质干细胞条件培养基联合白藜芦醇对人滋养层细胞的作用

目的:探讨人脐带间充质干细胞条件培养基联合白藜芦醇对人绒毛膜外滋养层细胞凋亡的影响。方法:通过CCK8细胞活力检测试剂盒测定白藜芦醇及其与人脐带间充质干细胞条件培养基共同处理人绒毛膜外滋养层细胞HTR8后对细胞增殖及活性的影响;细胞迁移试验检测白藜芦醇和人脐带间充质干细胞条件培养基对细胞迁移能力的影响;显微镜观察细胞形态,并用流式细胞仪检测细胞凋亡率的变化;Western blot检测白藜芦醇和人脐带间充质干细胞条件培养基对细胞凋亡相关蛋白Bax、Bcl-2以及迁移相关蛋白MMP-9表达的影响。结果:白藜芦醇能够抑制HTR8细胞增殖,抑制细胞迁移及MMP-9蛋白的表达,改变Bax和Bcl-2蛋白表达诱导细胞凋亡的作用。而人脐带间充质干细胞条件培养基能够逆转白藜芦醇对细胞的抑制作用。结论:人脐带间充质干细胞条件培养基能够通过调控Bax、Bcl-2、MMP-9的蛋白表达逆转白藜芦醇对人绒毛膜外滋养层细胞的抑制作用。人脐带间充质干细胞条件培养基可作为潜在的治疗人绒毛膜外滋养层细胞功能障碍的临床手段,孕妇需要小心使用白藜芦醇。     

Mesenchymal to embryonic incomplete transition of human cells by chimeric OCT4/3 (POU5F1) with physiological co-activator EWS

POU5F1 (more commonly known as OCT4/3) is one of the stem cell markers, and affects direction of differentiation in embryonic stem cells. To investigate whether cells of mesenchymal origin acquire embryonic phenotypes, we generated human cells of mesodermal origin with overexpression of the chimeric OCT4/3 gene with physiological co-activator EWS (product of the EWSR1 gene), which is driven by the potent EWS promoter by translocation. The cells expressed embryonic stem cell genes such as NANOG, lost mesenchymal phenotypes, and exhibited embryonal stem cell-like alveolar structures when implanted into the subcutaneous tissue of immunodeficient mice. Hierarchical analysis by microchip analysis and cell surface analysis revealed that the cells are subcategorized into the group of human embryonic stem cells and embryonal carcinoma cells. These results imply that cells of mesenchymal origin can be traced back to cells of embryonic phenotype by the OCT4/3 gene in collaboration with the potent cis-regulatory element and the fused co-activator. The cells generated in this study with overexpression of chimeric OCT4/3 provide us with insight into cell plasticity involving OCT4/3 that is essential for embryonic cell maintenance, and the complexity required for changing cellular identity.     

Stem cell‐based therapy for Huntington's disease

Huntington's disease (HD) is a late‐onset neurodegenerative disease characterized by a progressive loss of medium spiny neurons in the basal ganglia. The development of stem cell‐based therapies for HD aims to replace lost neurons and/or to prevent cell death. This review will discuss pre‐clinical studies which have utilized stem or progenitor cells for transplantation therapy using HD animal models. In several studies, neural stem and progenitor cells used as allotransplants and xenografts have been shown to be capable of surviving transplantation and differentiating into mature GABAergic neurons, resulting in behavioral improvements. Beneficial effects have also been reported for transplantation of stem cells derived from non‐neural tissue, for example, mesenchymal‐ and adipose‐derived stem cells, which have mainly been attributed to their secretion of growth and neurotrophic factors. Finally, we review studies using stem cells genetically engineered to over‐express defined neurotrophic factors. While these studies prove the potential of stem cells for transplantation therapy in HD, it also becomes clear that technical and ethical issues regarding the availability of stem cells must be solved before human trials can be conducted. J. Cell. Biochem. 114: 754–763, 2013. © 2012 Wiley Periodicals, Inc.     

Differentiation of human umbilical cord Wharton's jelly‐derived mesenchymal stem cells into germ‐like cells in vitro

Recent studies have demonstrated that mesenchymal stem cells could differentiate into germ cells under appropriate conditions. We sought to determine whether human umbilical cord Wharton's jelly‐derived mesenchymal stem cells (HUMSCs) could form germ cells in vitro. HUMSCs were induced to differentiate into germ cells in all‐trans retinoic acid, testosterone and testicular‐cell‐conditioned medium prepared from newborn male mouse testes. HUMSCs formed “tadpole‐like” cells after induction with different reagents and showed both mRNA and protein expression of germ‐cell‐specific markers Oct4 (POUF5), Ckit, CD49_f (α6), Stella (DDPA3), and Vasa (DDX4). Our results may provide a new route for reproductive therapy involving HUMSCs and a novel in vitro model to investigate the molecular mechanisms that regulate the development of the mammalian germ lineage. J. Cell. Biochem. 109: 747–754, 2010. © 2010 Wiley‐Liss, Inc.     

Polyethylenimine‐mediated gene delivery into human bone marrow mesenchymal stem cells from patients

Transplantation of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for post‐infarction left ventricular (LV) dysfunction. However, age‐related functional decline of stem cells has restricted their clinical benefits after transplantation into the infarcted myocardium. The limitations imposed on patient cells could be addressed by genetic modification of stem cells. This study was designed to improve our understanding of genetic modification of human bone marrow derived mesenchymal stem cells (hMSCs) by polyethylenimine (PEI, branched with Mw 25 kD), one of non‐viral vectors that show promise in stem cell genetic modification, in the context of cardiac regeneration for patients. We optimized the PEI‐mediated reporter gene transfection into hMSCs, evaluated whether transfection efficiency is associated with gender or age of the cell donors, analysed the influence of cell cycle on transfection and investigated the transfer of therapeutic vascular endothelial growth factor gene (VEGF). hMSCs were isolated from patients with cardiovascular disease aged from 41 to 85 years. Optimization of gene delivery to hMSCs was carried out based on the particle size of the PEI/DNA complexes, N/P ratio of complexes, DNA dosage and cell viability. The highest efficiency with the cell viability near 60% was achieved at N/P ratio 2 and 6.0 μg DNA/cm². The average transfection efficiency for all tested samples, middle‐age group (<65 years), old‐age group (>65 years), female group and male group was 4.32%, 3.85%, 4.52%, 4.14% and 4.38%, respectively. The transfection efficiency did not show any correlation either with the age or the gender of the donors. Statistically, there were two subpopulations in the donors; and transfection efficiency in each subpopulation was linearly related to the cell percentage in S phase. No significant phenotypic differences were observed between these two subpopulations. Furthermore, PEI‐mediated therapeutic gene VEGF transfer could significantly enhance the expression level.