Therapeutic potential of the immunomodulatory activities of adult mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) include a select population of resident cells within adult tissues, which retain the ability to differentiate along several tissue‐specific lineages under defined media conditions and have finite expansion potential in vitro. These adult progenitor populations have been identified in various tissues, but it remains unclear exactly what role both transplanted and native MSCs play in processes of disease and regeneration. Interestingly, increasing evidence reveals a unique antiinflammatory immunomodulatory phenotype shared among this population, lending support to the idea that MSCs play a central role in early tissue remodeling responses where a controlled inflammatory response is required. However, additional evidence suggests that MSCs may not retain infinite immune privilege and that the context with which these cells are introduced in vivo may influence their immune phenotype. Therefore, understanding this dynamic microenvironment in which MSCs participate in complex feedback loops acting upon and being influenced by a plethora of secreted cytokines, extracellular matrix molecules, and fragments will be critical to elucidating the role of MSCs in the intertwined processes of immunomodulation and tissue repair. Birth Defects Research (Part C) 90:67–74, 2010. © 2010 Wiley‐Liss, Inc.     

Impaired immunomodulatory capacity in adipose tissue-derived mesenchymal stem/stromal cells isolated from obese patients

Immune-modulatory properties of adipose tissue-derived mesenchymal stem/stromal cells (MSCs) might be susceptible to metabolic disturbances. We hypothesized that the immune-modulatory function of MSCs might be blunted in obese human subjects. MSCs were collected from abdominal subcutaneous fat of obese and lean subjects during bariatric or kidney donation surgeries, respectively. MSCs were co-cultured in vitro for 24 h with M1 macrophages, which were determined as M1or M2 phenotypes by flow cytometry, and cytokines measured in conditioned media. In vivo, lean or obese MSCs (5 × 10⁵), or PBS, were injected into mice two weeks after unilateral renal artery stenosis (RAS) or sham surgeries (n = 6 each). Fourteen days later, kidneys were harvested and stained with M1 or M2 markers. Lean MSCs decreased macrophages M1 marker intensity, which remained elevated in macrophages co-cultured with obese MSCs. TNF-α levels were four-fold higher in conditioned media collected from obese than from lean MSCs. RAS mouse kidneys were shrunk and showed increased M1 macrophage numbers and inflammatory cytokine expression compared with normal kidneys. Lean MSCs decreased M1 macrophages, M1/M2 ratio and inflammation in RAS kidneys, whereas obese MSCs did not. MSCs isolated from lean human subjects decrease inflammatory M1 macrophages both in vivo and in vitro, an immune-modulatory function which is blunted in MSCs isolated from obese subjects.     

Potential surrogate quantitative immunomodulatory potency assay for monitoring human umbilical cord-derived mesenchymal stem cells production

Mesenchymal stem cells (MSCs) play an important role as immune modulator through interaction with several immune cells, including macrophages. In this study, the immunomodulatory potency of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) was demonstrated in the in vivo middle cerebral artery occlusion (MCAo)-induced brain injury rat model and in vitro THP-1-derived macrophages model. At 24 h after induction of MCAo, hUC-MSCs was administered via tail vein as a single dose. Remarkably, hUC-MSCs could inhibit M1 polarization and promote M2 polarization of microglia in vivo after 14 days induction of MCAo. Compared with THP-1-derived macrophages which had been stimulated by lipopolysaccharide, the secretion of proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interferon-γ inducible protein (IP-10), were significantly reduced in the presence of hUC-MSCs. Moreover, the secretion of anti-inflammatory cytokine, interleukin-10 (IL-10), was significantly increased after cocultured with hUC-MSCs. Prostaglandins E2 (PGE2), secreted by hUC-MSCs, is one of the crucial immunomodulatory factors and could be inhibited in the presence of COX2 inhibitor, NS-398. PGE2 inhibition suppressed hUC-MSCs immunomodulatory capability, which was restored after addition of synthetic PGE2, establishing the minimum amount of PGE2 required for immunomodulation. In conclusion, our data suggested that PGE2 is a crucial potency marker involved in the therapeutic activity of hUC-MSCs through macrophages immune response modulation and cytokines regulation. This study provides the model for the development of a surrogate quantitative potency assay of immunomodulation in stem cells production.     

The effect of Trimetazidine and Diazoxide on immunomodulatory activity of human embryonic stem cell-derived mesenchymal stem cell secretome

Comprehensive proteome profiling of the factors secreted by mesenchymal stem cells (MSCs), referred to as secretome, revealed that it consists of cytokines, chemokines, growth factors, extracellular matrix proteins, and components of regeneration, vascularization, and hematopoiesis pathways. Harnessing this MSC secretome for therapeutic applications requires the optimization of production of secretary molecules. A variety of preconditioning methods have been introduced, which subject cells to stimulatory molecules to create the preferred response and stimulate persistent effects. Pharmacological preconditioning uses small molecules and drugs to increase survival of MSCs after transplantation or prolong release of effective secretary factors such as cytokines that improve immune system responses. In this study, we investigated the effect of secretome of human embryonic-derived mesenchymal stem cells (hESC-MSCs) preconditioned with Trimetazidine (TMZ) and Diazoxide (DZ) on immunomodulatory efficiency of these cells in LPS-induced peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human peripheral blood and treated with concentrated hESC-MSC-derived conditioned medium and then, the secreted levels of IL-10, TNFα and IL-1β were assessed by ELISA after induction with LPS. The results showed that TMZ and DZ-conditioned medium significantly enhanced immunomodulatory potential of hESC-MSCs by increasing the secretion of IL-10, TNFα and IL-1β from LPS- induced PBMCs. We also found that hESC-MSCs did not secrete mentioned cytokines prior to or after the preconditioning with TMZ and DZ. In conclusion, our results implied that TMZ and DZ can be used to promote the immunomodulatory effects of hESC-MSC secretome. It is obvious that for applying of these findings in clinical demands, the potency of different pre-conditioned MSCs secretome on immune response needs to be more clarified.     

骨髓间充质干细胞的免疫学研究进展

间充质干细胞（mesenchymal stem cells,MSCs）是骨髓中除造血干细胞以外的另一种成体干细胞,广泛分布于动物体内骨髓、肝脏、脂肪等多种组织中。MSCS具有强大的自我更新能力和多向分化潜能,是移植领域应用前景广阔的再生来源细胞;同时,MSCs是一种重要的免疫调节细胞,MSCs在炎症细胞因子刺激后对免疫系统表现出很强的抑制作用,所以MSCs有望应用于减少免疫排斥,延长移植物存活时间,治疗相关免疫失调症,如自身免疫疾病等方面。本文主要对间充质干细胞与免疫系统相互作用的研究做相关介绍。     

Trafficking and differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a heterogeneous population of stem/progenitor cells with pluripotent capacity to differentiate into mesodermal and non‐mesodermal cell lineages, including osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes, fibroblasts, myofibroblasts, epithelial cells, and neurons. MSCs reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues. When stimulated by specific signals, these cells can be released from their niche in the bone marrow into circulation and recruited to the target tissues where they undergo in situ differentiation and contribute to tissue regeneration and homeostasis. Several characteristics of MSCs, such as the potential to differentiate into multiple lineages and the ability to be expanded ex vivo while retaining their original lineage differentiation commitment, make these cells very interesting targets for potential therapeutic use in regenerative medicine and tissue engineering. The feasibility for transplantation of primary or engineered MSCs as cell‐based therapy has been demonstrated. In this review, we summarize the current knowledge on the signals that control trafficking and differentiation of MSCs. J. Cell. Biochem. 106: 984–991, 2009. © 2009 Wiley‐Liss, Inc.     

Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources

Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.     

Treatment of osteoarthritis with mesenchymal stem cells

Osteoarthritis(OA)is one of the most prevalent joint diseases with prominent symptoms affecting the daily life of millions of middle aged and elderly people.Despite this,there are no successful medical interventions that can prevent the progressive destruction of OA joints.The onset of pathological changes in OA is associated with deviant activity of mesenchymal stem cells(MSCs),the multipotent precursors of connective tissue cells that reside in joints.Current therapies for OA have resulted in poor clinical outcomes without repairing the damaged cartilage.Intra-articular delivery of culture-expanded MSCs has opened new avenues of OA treatment.Pre-clinical and clinical trials demonstrated the feasibility,safety,and efficacy of MSC therapy.The Wnt/β-catenin,bone morphogenetic protein 2,Indian hedgehog,and Mitogen-activated protein kinase signaling pathways have been demonstrated to be involved in OA and the mechanism of action of MSC therapies.     

骨髓间充质干细胞向角膜上皮细胞分化及其免疫调节作用

目的：探讨大鼠骨髓间充质干细胞（rBMMSCs）转分化为角膜上皮的潜能,并在体外共培养体系中研究rBMMSCs对促炎细胞因子干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）刺激下的人角膜上皮细胞（hCECs）的免疫调节作用。方法采用聚蔗糖梯密度离心法获得rBMMSCs,并通过上皮细胞培养微环境来诱导rBMMSCs分化为上皮样细胞。通过免疫组织化学方法鉴定CD29、CD34、CK5＆8和ZO-1等标记物在rBMMSCs及诱导的上皮样细胞中的表达。流式细胞术用来分析CD29/CD34的表达及细胞分化过程中表达量的变化。hCECs单独培养或与rBMMSCs共培养,并采用IFN-γ/TNF-α刺激24或48 h。通过流式细胞术来分析细胞间黏附分子-1（ICAM-1）于IFN-γ/TNF-α刺激前后在hCECs上的表达,并通过黏附分析实验验证rBMMSC条件培养基对单核细胞黏附于IFN-γ/TNF-α刺激后的hCECs的作用。多组间比较采用单因素方差分析（ANOVA）,两组间比较采用双侧t检验。结果成功分离rBMMSCs,细胞表达CD29,但不表达CD34。在上皮细胞培养条件中培养5 d,大约4﹪的rBMMSCs可分化为上皮样细胞。此类细胞失去了CD29的标志,转为表达CK5＆8和ZO-1。IFN-γ/TNF-α能显著上调hCECs中ICAM-1的表达,在IFN-γ/TNF-α处理24 h和48 h后,ICAM-1分别呈现10倍和8倍的升高,分别达到4524±554.2和3107±329.6（P=0.0025,0.0014）。但与MSC共同培养时,上调作用被显著抑制,ICAM-1平均值为1356±325.6（24 h）与1323±106.6（48 h）（P=0.0079,0.0024）。MSC条件培养基可显著抑制单核细胞对hCECs的黏附作用,黏附细胞数从（10.01±3.01）×10^3/ml细胞降至（2.21±0.19）×10^3/ml细胞（P=0.0271）。结论rBMMSCs可转分化为角膜上皮样细胞,并抑制由促炎细胞因子诱导的ICAM-1在hCECs上的表达,同时对促炎细胞因子诱导的单核细胞的黏附性具有抑制作用,提示BMMSCs具有在角膜炎症疾病和损伤修复中的治疗潜能。     

Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

骨髓间充质干细胞向神经细胞分化的研究

无论是在体外实验、还是在体内实验,MSCs都可以向中枢神经系统(CNS)神经细胞分化,但争议颇多。因为功能性神经元不仅要具有典型神经元的形态、特异性标记,还要求具有可兴奋性、能和其他神经元形成突触联系、产生突触电位等,所以对于骨髓间充质干细胞是否能诱导出真正具有功能的神经元存在很大分歧。在此对MSCs向神经细胞诱导分化研究的现况、存在的问题及发展前景给以综述。     

Immunogenicity of allogeneic mesenchymal stem cells

Mesenchymal stem cells (MSCs) inhibit proliferation of allogeneic T cells and express low levels of major histocompatibility complex class I (MHCI), MHCII and vascular adhesion molecule-1 (VCAM-1). We investigated whether their immunosuppressive properties and low immunophenotype protect allogeneic rat MSCs against cytotoxic lysis in vitro and result in a reduced immune response in vivo. Rat MSCs were partially protected against alloantigen-specific cytotoxic T cells in vitro. However, after treatment with IFN-γ and IL-1β, MSCs upregulated MHCI, MHCII and VCAM-1, and cytotoxic lysis was significantly increased. In vivo, allogeneic T cells but not allogeneic MSCs induced upregulation of the activation markers CD25 and CD71 as well as downregulation of CD62L on CD4(+) T cells from recipient rats. However, intravenous injection of allo-MSCs in rats led to the formation of alloantibodies with the capacity to facilitate complement-mediated lysis, although IgM levels were markedly decreased compared with animals that received T cells. The allo-MSC induced immune response was sufficient to lead to significantly reduced survival of subsequently injected allo-MSCs. Interestingly, no increased immunogenicity of IFN-γ stimulated allo-MSCs was observed in vivo. Both the loss of protection against cytotoxic lysis under inflammatory conditions and the induction of complement-activating antibodies will likely impact the utility of allogeneic MSCs for therapeutic applications.     

bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering.     

间充质干细胞在动脉粥样硬化治疗中的研究进展

动脉粥样硬化是一种病因复杂的血管壁慢性炎症性疾病。动脉粥样硬化及其相关并发症已成为人类死亡的主要原因,然而,其病因和发病机制尚未完全阐明,治疗效果还不满意。目前已经证实,动脉内皮细胞功能发生障碍是动脉粥样硬化的始动过程,内皮细胞功能失调和内皮细胞丢失是动脉粥样硬化症的主要特点;而血管平滑肌细胞的异常增生在动脉粥样硬化的发生发展中也扮演着重要角色。因此,探索有效措施促进有益的内皮细胞再生并抑制平滑肌细胞增生是血管损伤防治的关键。近年来有研究发现,体外输注的间充质干细胞能够向受损部位募集,并进一步分化为内皮细胞,修复损伤血管。然而,也有研究显示体外输注的间充质干细胞还可以分化为血管平滑肌细胞进而在血管局部增生,参与血管再狭窄的发生。文中综述了间充质干细胞输注对动脉粥样硬化发展的最新研究进展,希望为后续开展的用间充质干细胞治疗动脉粥样硬化的研究提供一定的参考。     

Inhibitory effect of mesenchymal stem cells on lymphocyte proliferation

We investigated the mechanism underlying the inhibitory effect of rat mesenchymal stem cells (MSCs) on non‐specific mitogen‐stimulated lymphocytes (LCs) and lymphoblasts (LBs). We used MSCs of passages 2–8 prepared from Sprague–Dawley (SD) rats. LCs were isolated from the spleens of SD rats. Mixed LCs reactions of mitomycin C‐treated MSCs with concanavalin A (ConA)‐stimulated LCs or LBs were performed, and the proliferation inhibition effect was tested by MTS assay. The cytotoxicity of MSCs against naïve and ConA‐stimulated LBs was detected, after co‐culturing for 24 h, by lactate dehydrogenase release assay. The rate of apoptosis of ConA‐stimulated LBs was measured by flow cytometry after incubation with MSCs for 9 h in the ratio 10:1. The MSCs were treated with Fas ligand (FasL), transforming growth factor (TGF)‐β, and interleukin (IL)‐10 blocking antibodies and co‐cultured with ConA‐stimulated LBs to observe the apoptosis and growth inhibitory effect. The main outcomes were bone marrow‐derived adherent CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD95⁺, and SH‐2⁺ MSCs. FasL, TGF‐β, and IL‐10 production by MSCs were visualized by immunocytochemical analysis. MSCs exhibited a dose‐dependent growth inhibitory effect on ConA‐stimulated LCs and LBs. When treated with anti‐FasL and anti‐IL‐10 blocking antibodies, the inhibitory effect of MSCs on LBs proliferation, and the effect of apoptosis induction on LBs decreased. Anti‐TGF‐β blocking antibody treatment did not significantly influence MSCs. Therefore, the inhibitory effects of MSCs against activated LBs were significantly stronger than that against naïve LCs. FasL and IL‐10, rather than TGF‐β, play important roles in the immunosuppressive effects of MSCs. Copyright © 2008 John Wiley & Sons, Ltd.     

Comparative characterization of mesenchymal stem cells from different age groups of cynomolgus monkeys

Bone marrow mesenchymal stem cells(BM-MSCs) are a potential tool for cell therapy and tissue engineering.In this study,we carried on a comparative study of the characteristics of MSCs from different age cynomolgus monkeys.A variety of factors,including donor age,must be considered before further applications,and various tests should be used to properly assess MSCs before the clinical application,especially when a prolonged culture and ex vivo expansion is necessary.     

Comparative studies of different cryopreservation methods for mesenchymal stem cells derived from human fetal liver

Fetal stem cells possess some intriguing characteristics, which delineate them as promising cellular therapeutics. They are less immunogenic, at lower stage of differentiation and have higher potential for repopulation and migration. Furthermore, the fetal stem cells secrete a set of cytokines and growth factors, which stimulate the regeneration of the recipient tissue. The present study indicated that the adhesive fraction of human fetal liver cells possessed the morphological characteristics of mesenchymal stem cells, as well as potential to differentiate into adipocyte and osteoblast lineages. The immunophenotypic analysis showed that the cells expressed CD13, CD73, CD90 and CD105 (typical for mesenchymal stem cells) and lacked the haematopoietic lineage markers CD34 and CD45. Addressing the issue of the low‐temperature storage of the human fetal liver cells, four different methods for cryopreservation were assessed: conventional slow freezing, program freezing and two vitrification protocols. The obtained results demonstrated that the cells were cryotolerant and maintained their properties and differentiation potential after thawing. Program freezing showed to be the most efficient method for cryopreservation of the investigated cells.     

脂肪间充质干细胞来源外泌体的功能

外泌体是细胞外膜质纳米囊泡,将蛋白质、核酸(DNA和RNA)转运到靶细胞中,介导局部和系统的细胞间通信,从而改变受体细胞的行为.这些小泡在许多生物功能中发挥重要作用,如脂肪合成、免疫调节、神经再生和肿瘤调节等.脂肪间充质干细胞目前被认为是细胞治疗和再生医学领域中一种功能丰富的工具,可产生和分泌多种外泌体,继承细胞的多种...     

Hypoxia enhances buffalo adipose-derived mesenchymal stem cells proliferation,stemness, and reprogramming into induced pluripotent stem cells

Adipose tissue-derived mesenchymal stem cells (ASCs) from livestock are valuable resources for animal reproduction and veterinary therapeutics. Previous studies have shown that hypoxic conditions were beneficial in maintaining the physiological activities of ASCs. However, the effects of hypoxia on buffalo ASCs (bASCs) remain unclear. In this study, the effects of hypoxia on proliferation, stemness, and reprogramming into induced pluripotent stem cells (iPSCs) of bASCs were examined. The results showed that the hypoxic culture conditions (5% oxygen) enhanced the proliferation and colony formation of bASCs. The expression levels of proliferation-related genes, and secretion of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were significantly enhanced in hypoxia. Hypoxic culture conditions activated hypoxia-inducible factor-1α (HIF-1α), thereby contributing to the secretion of bFGF and VEGF, which in turn enhanced the expression of HIF-1α and promoted the proliferation of bASCs. Furthermore, in hypoxic culture conditions, bASCs exhibited the main characteristics of mesenchymal stem cells, and the expression levels of the pluripotent markers OCT4, NANOG, C-MYC, and the differentiation capacity of bASCs were significantly enhanced. Finally, bASCs were more efficiently and easily reprogrammed into iPSCs in hypoxic culture conditions and these iPSCs exhibited some characteristics of naïve pluripotent stem cells. These findings provide the theoretical guidance for elucidating the detailed mechanism of hypoxia on physiological activities of bASCs including proliferation, stemness maintenance, and reprogramming.     

1-磷酸鞘氨醇促进间充质干细胞向心肌分化

为研究1-磷酸鞘氨醇 (Sphingosine-1-phosphate,S1P) 对脐带间充质干细胞 (Umbilical cord mesenchymal stem cells,UC-MSCs) 和脂肪间充质干细胞 (Adipose derived mesenchymal stem cells,AD-MSCs) 向心肌分化的影响,探索其适宜的作用时间和浓度,将UC-MSCs和AD-MSCs接种到培养板,用添加不同浓度S1P的心肌细胞培养液诱导两种干细胞向心肌分化,诱导时间分为7 d、14 d和28 d。采用免疫荧光染色检测心肌特异性蛋白,α-肌动蛋白 (α-actin)、缝隙连接蛋白 (Connexin-43) 以及肌球蛋白重链 (MYH-6) 的表达,并通过共聚焦显微镜和荧光显微镜进行观察;采用MTT分析细胞的活性;膜片钳检测分化细胞的钙瞬变 (此为心肌细胞的功能性指标)。结果表明,S1P与心肌细胞培养液协同作用,能够促进UC-MSCs和AD-MSCs向心肌细胞的分化。并且,随着S1P浓度的增加,促分化作用增强,但细胞活性降低。S1P在心肌细胞培养液中的适宜作用时间为14 d,适宜作用浓度为0.5 μmol/L。而且联合心肌细胞培养液可以使UC-MSCs和AD-MSCs的心肌分化细胞产生钙瞬变,具有类似心肌细胞的功能性。S1P能够与心肌细胞培养液协同作用,促进UC-MSCs和AD-MSCs的心肌功能性分化。