骨髓间充质干细胞向肝（样）细胞的分化及其在肝脏疾病的应用

骨髓间充质干细胞在肝脏病理条件或体外诱导分化为有功能的肝（样）细胞以及对受损肝脏的修复作用是目前研究的热点。本文就骨髓间充质干细胞分化潜能及其可能的机制、免疫学特性以及在肝脏疾病的应用研究作系统的综述。     

Biological Characteristics and Odontogenic Differentiation Effects of Calcium Silicate-Based Pulp Capping Materials

We compared calcium silicate-based pulp capping materials to conventional calcium hydroxide in terms of their biological properties and potential effects on odontogenic differentiation in human dental pulp stem cells (hDPSCs). We cultured hDPSCs on disks (7-mm diameter, 4-mm high) of ProRoot MTA (Dentsply Tulsa Dental Specialties), Biodentine (Septodont), TheraCal LC (Bisco), or Dycal (Dentsply Tulsa Dental Specialties). Cell viability was assessed with cell counting (CCK) and scanning electron microscopy (SEM). Odontogenic activity was assessed by measuring alkaline phosphatase (ALP) activity and gene expression (quantitative real-time PCR). CCK assays showed that Dycal reduced cell viability compared to the other materials (p < 0.05). SEM showed low and absent cell attachment on TheraCal LC and Dycal disks, respectively. hDPSCs exposed to TheraCal LC and Dycal showed higher ALP activity on day 6 than the control group (p < 0.05). The day-9 Runx2 expression was higher in the ProRoot MTA and TheraCal LC groups than in the control group (p < 0.05). On day 14, the ProRoot MTA group showed the highest dentin sialophosphoprotein levels (not significant; p > 0.05). In conclusion, various pulp capping materials, except Dycal, exhibited biological properties favorable to hDPSC viability. ProRoot MTA and TheraCal LC promoted higher Runx2 expression than Biodentine. Future studies should explore the odontogenic potential of pulp capping materials.     

Mesenchymal Stem Cells Derived from Bone Marrow of Diabetic Patients Portrait Unique Markers Influenced by the Diabetic Microenvironment

Cellular microenvironment is known to play a critical role in the maintenance of human bone marrow-derived mesenchymal stem cells (BM-MSCs). It was uncertain whether BM-MSCs obtained from a ''diabetic milieu'' (dBM-MSCs) offer the same regenerative potential as those obtained from healthy (non-diabetic) individuals (hBM-MSCs). To investigate the effect of diabetic microenvironment on human BM-MSCs, we isolated and characterized these cells from diabetic patients (dBM-MSCs). We found that dBM-MSCs expressed mesenchymal markers such as vimentin, smooth muscle actin, nestin, fibronectin, CD29, CD44, CD73, CD90, and CD105. These cells also exhibited multilineage differentiation potential, as evident from the generation of adipocytes, osteocytes, and chondrocytes when exposed to lineage specific differentiation media. Although the cells were similar to hBM-MSCs, 6% (3/54) of dBM-MSCs expressed proinsulin/C-peptide. Emanating from the diabetic microenvironmental milieu, we analyzed whether in vitro reprogramming could afford the maturation of the islet-like clusters (ICAs) derived from dBM-MSCs. Upon mimicking the diabetic hyperglycemic niche and the supplementation of fetal pancreatic extract, to differentiate dBM-MSCs into pancreatic lineage in vitro, we observed rapid differentiation and maturation of dBM-MSCs into islet-like cell aggregates. Thus, our study demonstrated that diabetic hyperglycemic microenvironmental milieu plays a major role in inducing the differentiation of human BM-MSCs in vivo and in vitro.     

Role of bone marrow-derived mesenchymal stem cell defects in CD8+CD28– suppressor T-lymphocyte induction in patients with immune thrombocytopenia and associated mechanisms

Many immune dysfunctions participate in immune thrombocytopenia (ITP) pathogenesis, including numeric and functional defects in suppressor T (Ts) cells and immune-regulation abnormalities in mesenchymal stem cells (MSCs). Recent studies showed that MSCs can promote Ts cell differentiation. Thus, we compared the Ts cell induction ability of bone marrow-derived MSCs (BM-MSCs) between patients with ITP and normal controls (NCs), and examined the mechanism of this difference. Co-culture of CD8⁺ T cells with BM-MSCs revealed that BM-MSCs elevated Ts cell percentage and function, but the efficiency was lower in patients with ITP than in NCs. Blockade experiments showed that blockade of interleukin 6 (IL-6) partially reversed Ts cell induction by BM-MSCs. Addition of exogenous IL-6 down-regulated Ts cell apoptosis. Moreover, BM-MSCs enhanced IL-10 secretion and inhibition ability of Ts cells. IL-6 secretion, regulatory abilities of IL-10 expression in Ts cells, and the enhanced efficiency of Ts cells inhibition function by BM-MSCs were all decreased in patients with ITP. All-trans retinoic acid preconditioning promoted BM-MSC induction of Ts cell percentages and umbilical cord-derived (UC) MSCs efficiently improved ITP Ts cell numbers and dysfunction. In conclusion, defects of BM-MSCs in Ts cell induction are involved in ITP pathogenesis, and exogenous UC-MSCs may be useful for ITP therapy.     

Decellularized ovine arteries as biomatrix scaffold support endothelial of mesenchymal stem cells

The differentiation rate of adipose-derived mesenchymal stem cells (Ad-MSCs) into endothelial cells is always lower under normal condition, which limits further clinical application of Ad-MSCs for angiogenesis regenerative medicine and needs to be enhanced. In the present study, the tissue-specific-derived decellularized ovine arteries matrix (DCS) was used as scaffold to investigate the pro-endothelial differentiation ability of decellularized ovine arteries matrix as well as the underlying mechanisms. The prepared decellularized ovine arteries matrix by the combination of enzymatic and chemical decellularization approaches preserved macroscopic 3D architecture, native composition and ultrastructure of natural ovine arteries. The RT-PCR, histopathological and immunofluorescence assay results suggested that DCS could increase the proliferation ability of MSC. What’s more, the DCS could also induce the endothelial differentiation of MSC, which was further enhanced by adding VEGF. Our results showed that natural 3D matrix from decellularized ovine arteries could induce the endothelial differentiation of AD-MSCs alone or with the combination of VEGF. Our results indicated that the decellularized ovine arteries matrix would serve as an efficient culture system for promoting endothelial differentiation of Ad-MSCs.     

Noggin inhibits chondrogenic but not osteogenic differentiation in mesodermal stem cell line C1 and skeletal cells

Osteoblasts and chondroblasts are derived from common mesenchymal progenitors. Although bone morphogenetic protein induces mesenchymal differentiation into both osteogenic and chodrogenic lineage cells in vitro, its inhibitor, Noggin, is expressed exclusively during chondrogenic but not osteogenic differentiation in an embryonal carcinoma-derived mesodermal cell line, C1. We hypothesized that Noggin may regulate cell differentiation in a lineage-specific manner. To test this hypothesis, Noggin was overexpressed using recombinant adenovirus (Ad/Noggin) in mesodermal C1 cells to examine whether Noggin specifically inhibits chondrogenic differentiation. Noggin overexpression by recombinant adenovirus infection reduced Sox9, patched, Ihh, and type II, X, and XI collagen mRNA expression levels in C1 cell aggregates that were induced to differentiate into chondrocyte lineage by culturing in differentiation medium. In contrast, Noggin overexpression did not affect osteogenic differentiation in C1 cells because osteoblast phenotypic markers such as osteocalcin and alkaline phosphatase mRNA levels were not altered. We further examined whether Noggin also differentially affects chondrogenesis and osteogenesis in limb development by using organ cultures of long bone. Ad/Noggin infection into 15.5 d post conception limb skeletal rudiments that were cultured on filter membrane in vitro or on the chorioallantoic membranes in ovo inhibited the levels of chondrogenesis, which were evaluated based on alcian blue staining. These results suggest that Noggin specifically blocks chondrogenic differentiation, rather than osteogenic differentiation, in mesodermal stem cell line C1 and skeletal cells.     

自体骨髓间充质干细胞移植治疗心肌梗死的进展

心肌梗死可引起心肌细胞的丢失,进而引起心脏功能的下降,是心力衰竭最主要的病因。骨髓间充质干细胞（BM—MSCs）是一类具有横向分化为各系统器官和组织能力的干细胞,能补充丢失的心肌细胞并通过旁分泌等机制增强心功能,为心肌梗死提供一种全新的治疗方法,极具发展潜力。本文对BM—MSCs的分离培养、诱导因素、移植以及临床研究等方面进行了综述。     

骨髓间充质干细胞治疗急性肝衰竭的研究进展

骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)是干细胞中一类具有自我增殖和分化潜能的细胞.在适宜的微环境下,BMSCs可分化为骨细胞、软骨细胞、脂肪细胞、神经细胞、肝细胞等多种细胞,基于此种特性使之成为原位肝移植或生物人工肝支持系统的新型种子细胞.本文就BMSCs的...     

Nanoparticles of Bioactive Glass Enhance Biodentine Bioactivity on Dental Pulp Stem Cells

This study aimed to investigate the cytotoxicity and bioactivity of a novel nanocomposite containing nanoparticles of bioactive glass (nBGs) on human dental pulp stem cells (hDPSCs). nBGs were synthesized by the sol–gel method. Biodentine (BD) nanocomposites (nBG/BD) were prepared with 2 and 5% wt of nBG content; unmodified BD and glass ionomer cement were used as references. Cell viability and attachment were evaluated after 3, 7 and 14 days. Odontogenic differentiation was assessed with alkaline phosphatase (ALP) activity after 7 and 14 days of exposure. Cells successfully adhered and proliferated on nBG/BD nanocomposites, cell viability of nanocomposites was comparable with unmodified BD and higher than GIC. nBG/BD nanocomposites were, particularly, more active to promote odontogenic differentiation, expressed as higher ALP activity of hDPSCs after 7 days of exposure, than neat BD or GIC. This novel nanocomposite biomaterial, nBG/BD, allowed hDPSC attachment and proliferation and increased the expression of ALP, upregulated in mineral-producing cells. These findings open opportunities to use nBG/BD in vital pulp therapies.     

内皮细胞特异性分子 1预处理对小鼠骨髓间充质干细胞生物学特性的影响

目的:探讨内皮细胞特异性分子 1（ESM 1）孵育预处理对小鼠骨髓间充质干细胞生物学特性的影响,为干细胞移植提供实验依据。方法: 培养及鉴定骨髓间充质干细胞。提取细胞数及浓度固定的初级培养液中的骨髓间充质干细胞,并分为两组:对照组（不进行预处理）及3个浓度（005 μg/ml、01 μg/ml、015 μg/ml）的ESM 1预处理组。每组设4个复孔,每孔均处理60 min。收集每孔中的条件培养液,检测预处理对骨髓间充质干细胞分泌血管内皮生长因子(VEGF)及碱性磷酸酶(ALP)的影响。结果: ESM 1孵育预处理骨髓间充质干细胞后,其存活率明显增加,而凋亡率显著下降,且骨髓间充质干细胞增殖能力随着ESM 1预处理浓度的增加而逐渐升高,凋亡率却呈相反的下降趋势。ESM 1预处理后,骨髓间充质干细胞分泌VEGF、ALP的水平明显升高,且随着ESM 1浓度的增加,骨髓间充质干细胞分泌VEGF、ALP的水平也相应增加。ESM 1孵育预处理后的骨髓间充质干细胞,细胞形态一致,生长及分化速度加快。结论: ESM 1孵育预处理骨髓间充质干细胞不仅促进骨髓间充质干细胞增殖及降低其凋亡,而且可增加VEGF、ALP的分泌。ESM 1通过活化骨髓间充质干细胞,提高细胞本身的潜能,继而提高其存活、增殖的能力, 为临床急性心肌梗死的治疗提供了广泛的应用前景。     

Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells

Like mesenchymal stem cells from bone marrow (BM-MSCs), adipose tissue-derived adult stem cells (ADAS cells) can differentiate into several lineages and present therapeutical potential for repairing damaged tissues. The use of allogenic stem cells can enlarge their therapeutical interest, provided that the grafted cells could be tolerated. We investigate here, for the first time, the immunosuppressive properties of ADAS cells compared with the well-characterized immunosuppressive properties of BM-MSCs. ADAS cells did not provoke in vitro alloreactivity of incompatible lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogens. The impairment of inhibition when ADAS cells and BM-MSCs were separated from lymphocytes by a permeable membrane suggests that cell contact is required for a full inhibitory effect. Hepatocyte growth factor is secreted by both stem cells but, similar to interleukin-10 and transforming growth factor-beta (TGF-beta), the levels of which were undetectable in supernatants of MLR inhibited by ADAS cells or BM-MSCs, it did not seem implicated in the stem cell suppressive effect. These findings support that ADAS cells share immunosuppressive properties with BM-MSCs. Therefore, ADAS cell-based reconstructive therapy could employ allogenic cells and because of their immunosuppressive properties, ADAS cells could be an alternative source to BM-MSCs to treat allogenic conflicts.     

体外诱导骨髓间充质干细胞向神经元样细胞分化的研究

目的观察人骨髓间充质干细胞（BM-MSCs）体外扩增和定向诱导分化为神经元样细胞的变化,为其临床应用奠定基础。方法采用密度梯度离心法分离BM-MSCs细胞,用MesencultTM培养基和贴壁培养法培养、纯化和扩增细胞,流式细胞术检测细胞表面CD29和CD90分子表达率。采用20 ng/ml重组碱性成纤维细胞生长因子（bFGF）、20 ng/ml新型人表皮生长因子（hEGF）和20 g/L二甲基亚砜（DMSO）、5 mmol/Lβ-巯基乙醇（BME）分别诱导BM-MSCs;采用免疫组化法检测细胞的神经元特异性烯醇化酶（NSE）、胶原纤维酸性蛋白（GFAP）、波形蛋白（VIM）表达情况。结果BM-MSCs细胞增殖迅速,3周可传代培养;BM-MSCs传十代扩增1－2×10^3倍。分离和扩增BM-MSCs CD29、CD90强表达率分别为95.02%、93.81%。药物诱导后的BM-MSCs发生轴突等神经元样细胞变化,阳性表达NSE、GFAP、VIM分子。结论人BM-MSCs能体外培养和扩增,并能定向诱导分化为神经元样细胞。     

Inhibitory effect of alcohol on osteogenic differentiation in human bone marrow-derived mesenchymal stem cells

BACKGROUND: Alcohol-induced osteoporosis is characterized by a considerable suppression of osteogenesis. The objective of this investigation was to determine the effect of alcohol on gene expression, protein synthesis, and mineralization in human bone marrow-derived mesenchymal stem cells induced toward osteogenic differentiation in vitro. METHODS: Human bone marrow-derived mesenchymal stem cells induced toward osteogenesis were cultured in the presence or absence of 50 mM alcohol. Stem cells were characterized by using SH2 antibody to the cell-surface antigen CD105/endoglin, and their proliferation in the presence of alcohol was quantified. The expression of genes for early, middle, and late markers of the osteogenic lineage was quantified by Northern analysis, and bone matrix protein synthesis was assayed. The effect of alcohol on cell-mediated matrix mineralization in terminally differentiated cultures was determined by von Kossa staining. RESULTS: Fluorescence-activated cell sorting analysis of human mesenchymal stem cells separated with a Percoll gradient proved 99% homogeneity by using SH2 antibody to the surface antigen CD105. Dose-dependent inhibition of proliferation of these stem cells occurred at concentrations greater than 50 mM alcohol. Gene expression of osteoblast-specific factor 2/core binding factor a1 (Osf2/Cbfa1), type I collagen, alkaline phosphatase, and osteocalcin (early, middle, and late markers for osteogenesis, respectively) was analyzed with and without osteogenic induction and treatment with 50 mM alcohol. After induction, Osf2/Cbfa1 levels were unresponsive to alcohol. To determine the effect of alcohol on human mesenchymal stem cell progression along the osteogenic pathway, messenger RNA (mRNA) levels for type I collagen, alkaline phosphatase, and osteocalcin were examined after osteogenic induction. After osteogenic induction, alcohol down-regulated the gene expression of type I collagen and significantly reduced its synthesis. Alcohol did not alter mRNA expression of alkaline phosphatase, a midstage marker for osteogenesis, but significantly decreased its activity compared with osteogenic induction alone. After induction, osteocalcin remained unchanged by alcohol at both the mRNA and protein levels. Histochemistry revealed decreased alkaline phosphatase staining and fewer alkaline phosphatase-positive cells in alcohol-treated human mesenchymal stem cell cultures. von Kossa staining revealed a reduction in the number of mineralizing nodules in stem cell cultures after alcohol treatment. CONCLUSIONS: Collectively, the data suggest that alcohol alters osteogenic differentiation in human bone marrow-derived mesenchymal stem cell cultures during lineage progression and provide further insight into alcohol-induced reduced bone formation.     

Structural investigation of donor age effect on human bone marrow mesenchymal stem cells: FTIR spectroscopy and imaging

Stem cell studies hold enormous potential for development of new therapies for tissue regeneration and repair. Bone marrow mesenchymal stem cells (BM-MSCs) can differentiate into a variety of non-hematopoietic tissues and contribute maintenance of healthy hematopoiesis by providing supportive cellular microenvironment into BM. Here, we investigated age-related differences in BM-MSCs by using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and FTIR imaging together with hierarchical clustering as a novel methods to clarify global alterations in the structure and function of macromolecules in characterized BM-MSCs of different aged donors. The results may contribute to identification of age-related new molecular marker(s) to determine the effects of donor age on MSCs. The spectral results reflected that there were significant increases in the concentration of saturated lipids, proteins, glycogen, and nucleic acids in children and adolescent group BM-MSCs when compared to the infants and early and mid adults. The concentration of mentioned macromolecules in adult (early and mid) BM-MSCs were significantly lower than the concentrations in the children and adolescents. These results were attributed to the increase in the proliferation activity in younger BM-MSCs. The distribution of macromolecules into the cells was shown as in the form of chemical maps by FTIR imaging, and the results are in agreement with the ATR-FTIR spectroscopy results. The cellular activity degree was determined by the thiazolyl blue tetrazolium bromide (MTT) proliferation assay to support ATR-FTIR spectroscopy results. BM-MSCs of five different age groups were discriminated by making the hierarchical cluster analysis where the spectral data according to alterations in structure and composition of macromolecules were considered.     

Total body irradiation tremendously impair the proliferation,differentiation and chromosomal integrity of bone marrow-derived mesenchymal stromal stem cells

Total body irradiation (TBI) is frequently used in hematopoietic stem cell transplantation (HSCT) and is associated with many complications due to radiation injury to the normal cells, including normal stem cells. Nevertheless, the effects of TBI on the mesenchymal stromal stem cell (MSC) are not fully understood. Bone marrow-derived MSCs (BM-MSCs) isolated from normal adults were irradiated with 200 cGy twice daily for consecutive 3 days, a regimen identical to that used in TBI-conditioning HSCT. The characteristics, differentiation potential, cytogenetics, hematopoiesis-supporting function, and carcinogenicity of the irradiated BM-MSCs were then compared to the non-irradiated control. The irradiated and non-irradiated MSCs shared similar morphology, phenotype, and hematopoiesis-supporting function. However, irradiated MSCs showed much lower proliferative and differentiative potential. Irradiation also induced clonal cytogenetic abnormalities of MSCs. Nevertheless, the carcinogenicity of irradiated MSCs is low in vitro and in vivo. In parallel with the ex vivo irradiation experiments, decreased proliferative and differentiative abilities and clonal cytogenetic abnormalities can also be found in MSCs isolated from transplant recipients who had received TBI-based conditioning previously. Thus, TBI used in HSCT drastically injury MSCs and may contribute to the development of some long-term complications associated with clonal cytogenetic abnormality and poor adipogenesis and osteogenesis after TBI.     

Bone marrow mesenchymal stromal cells (BM-MSCs) from healthy donors and auto-immune disease patients reduce the proliferation of autologous- and allogeneic-stimulated lymphocytes in vitro

OBJECTIVES: To investigate the ability of bone marrow (BM)-derived mesenchymal stromal cells (BM-MSCs) in suppressing the proliferation of stimulated lymphocytes across a range of conditions including autologous BM-MSCs derived from autoimmune disease (AD) patients. METHODS: In vitro cultures of BM-MSCs from healthy donors and AD patients were established and characterized by their differentiation potential into adipocytes and osteoblasts, and their fibroblast-colony-forming unit (CFU-F) ability and phenotype by flow cytometry. BM-MSCs (irradiated and non-irradiated) from healthy and AD patients were tested for their ability to suppress the in vitro proliferation of autologous and allogeneic peripheral blood mononuclear cells (PBMC) (from healthy donors and patients suffering from various ADs) stimulated with anti-CD3epsilon antibody alone or in combination with anti-CD28 antibody. The anti-proliferative effect of the BM-MSCs from healthy donors was tested also on transformed B-cell lines as a model of non-antigen-stimulated lymphocytes. RESULTS: BM-MSCs from healthy donors and AD patients reduced the proliferation of autologous and allogeneic PBMCs by up to 90% in a cell dose-dependent fashion. The immunosuppression was independent of the proliferation of the BM-MSCs and was also effective on already proliferating cells. It was independent also of the clinical activity of AD. An MSC dose-dependent pattern of suppression of proliferation was observed also with transformed B-cell lines, similar to that observed with proliferating PBMC. CONCLUSIONS: The BM-MSCs exhibit extensive anti-proliferative properties against lymphocytes under different conditions. This property might offer a form of immunomodulatory cellular therapy for AD patients if further confirmed in animal models.     

Mineral Trioxide Aggregate (MTA) Upregulates the Expression of DMP1 in Direct Pulp Capping in the Rat Molar

Mineral trioxide aggregate (MTA) is an alternative endodontic material that predicts conductive or inductive calcified tissue formation from immature pulp mesenchymal stem cells (IPMSCs). The purpose of this study was to investigate whether MTA could promote reparative odontoblast differentiation via IPMSCs in the early phase of regeneration and compare with calcium hydroxide (CH). Direct pulp capping using calcium hydroxide (CH), MTA, and MTA with platelet-rich plasma (MTA + PRP) was performed on maxillary first molars of 8-week-old male Wistar rats (n = 36). After 3, 7, or 14 days, the teeth were analyzed for mineral density (MD) and volume of MD (VMD) via micro-focusing computed tomography (µCT), nestin, dentin matrix acidic phosphoprotein 1 (DMP1) immunohistochemistry, and real-time PCR for DMP1 mRNA expression. MTA stimulated the early phase differentiation of the IPMSCs into odontoblasts, with positive results for nestin and DMP1 compared with CH. Moreover, MTA + PRP stimulated calcified granule and dentin bridge formation through calcium mineral deposition, following the induction of DMP1 mRNA expression in IPMSCs. Our results suggested that the combination of MTA and PRP is an effective and clinically applicable method for activating endogenous dental pulp stem cells into odontoblasts in the early stages of pulp regeneration.     

小鼠DNA酶真核表达质粒在小鼠骨髓间充质干细胞中的表达

目的 将构建好的小鼠DNA酶(DNaseI)真核表达质粒转染到小鼠骨髓间充质干细胞中,观察目的基因在细胞内的表达及分泌情况.方法 采用脂质体法将已构建好的小鼠DNaseI的真核表达质粒转染人小鼠骨髓间充质干细胞中,用Western blotting法检测DNaseI基因在骨髓间充质干细胞内的表达及DNA-甲基绿比色法检测培养上清液中DNaseI的活性.结果 小鼠DNaseI的真核表达质粒能够转染入小鼠骨髓间充质干细胞中,转染效率约为30%.转染后的小鼠骨髓间充质干细胞可以表达DNaseI蛋白且分泌出具有活性的DNaseI.结论 脂质体法能够将小鼠DNaseI的真核表达质粒转染入小鼠骨髓间充质干细胞中,小鼠DNaseI基因在小鼠骨髓间充质干细胞中成功表达并分泌具有生物活性的DNaseI.     

Delayed enrichment of mesenchymal cells promotes cardiac lineage and calcium transient development

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can be induced to differentiate into myogenic cells. Despite their potential, previous studies have not been successful in producing a high percentage of cardiac-like cells with a muscle phenotype. We hypothesized that cardiac lineage development in BM-MSC is related to cell passage, culture milieu, and enrichment for specific cell subtypes before and during differentiation. Our study demonstrated that Lin⁻ BM-MSC at an intermediate passage (IP; P8-P12) expressed cardiac troponin T (cTnT) after 21 days in culture. Cardiac TnT expression was similar whether IP cells were differentiated in media containing 5-azacytidine + 2% FBS (AZA; 14%) or 2% FBS alone (LS; 12%) and both were significantly higher than AZA + 5% FBS. This expression was potentiated by first enriching for CD117/Sca-1 cells followed by differentiation (AZA, 39% and LS, 28%). A second sequential enrichment for the dihydropyridine receptor subunit α2δ1 (DHPR-α2) resulted in cardiac TnT expressed in 54% of cultured cells compared to 28% of cells after CD117/Sca-1⁺ enrichment. Cells enriched for CD117/Sca-1 and subjected to differentiation displayed spontaneous intracellular Ca²⁺ transients with an increase in transient frequency and a 60% decrease in the transient duration amplitude between days 14 and 29. In conclusion, IP CD117/Sca-1⁺ murine BM-MSCs display robust cardiac muscle lineage development that can be induced independent of AZA but is diminished under higher serum concentrations. Furthermore, temporal changes in calcium kinetics commensurate with increased cTnT expression suggest progressive maturation of a cardiac muscle lineage. Enrichment with CD117/Sca-1 to establish lineage commitment followed by DHPR-α2 in lineage developing cells may enhance the therapeutic potential of these cells for transplantation.