Stem Cells International The latest articles from Hindawi Publishing Corporation © 2015 , Hindawi Publishing Corporation . All rights reserved. Dynamic Network-Based Relevance Score Reveals Essential Proteins and Functional Modules in Directed Differentiation Tue, 21 Apr 2015 13:11:30 +0000 The induction of stem cells toward a desired differentiation direction is required for the advancement of stem cell-based therapies. Despite successful demonstrations of the control of differentiation direction, the effective use of stem cell-based therapies suffers from a lack of systematic knowledge regarding the mechanisms underlying directed differentiation. Using dynamic modeling and the temporal microarray data of three differentiation stages, three dynamic protein-protein interaction networks were constructed. The interaction difference networks derived from the constructed networks systematically delineated the evolution of interaction variations and the underlying mechanisms. A proposed relevance score identified the essential components in the directed differentiation. Inspection of well-known proteins and functional modules in the directed differentiation showed the plausibility of the proposed relevance score, with the higher scores of several proteins and function modules indicating their essential roles in the directed differentiation. During the differentiation process, the proteins and functional modules with higher relevance scores also became more specific to the neuronal identity. Ultimately, the essential components revealed by the relevance scores may play a role in controlling the direction of differentiation. In addition, these components may serve as a starting point for understanding the systematic mechanisms of directed differentiation and for increasing the efficiency of stem cell-based therapies. Chia-Chou Wu, Che Lin, and Bor-Sen Chen Copyright © 2015 Chia-Chou Wu et al. All rights reserved. Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages Mon, 20 Apr 2015 11:18:07 +0000 The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson’s correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson’s correlation revealed high correlation () between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs. Won-Jae Lee, Ryoung-Hoon Jeon, Si-Jung Jang, Ji-Sung Park, Seung-Chan Lee, Raghavendra Baregundi Subbarao, Sung-Lim Lee, Bong-Wook Park, William Allan King, and Gyu-Jin Rho Copyright © 2015 Won-Jae Lee et al. All rights reserved. Rho/MRTF-A-Induced Integrin Expression Regulates Angiogenesis in Differentiated Multipotent Mesenchymal Stem Cells Wed, 08 Apr 2015 11:28:03 +0000 Mesenchymal stem cells (MSCs) are known to undergo endothelial differentiation in response to treatment with vascular endothelial growth factor (VEGF), but their angiogenic ability is poorly characterized. In the present study, we aimed to further investigate the role of Rho/MRTF-A in angiogenesis by MSCs and the effect of the Rho/MRTF-A pathway on the expression of integrins α1β1 and α5β1, which are known to mediate physiological and pathological angiogenesis. Our results showed that increased expression of α1, α5, and β1 was observed during angiogenesis of differentiated MSCs, and the Rho/MRTF-A signaling pathway was demonstrated to be involved in regulating the expression of integrins α1, α5, and β1. Luciferase reporter assay and ChIP assay determined that MRTF-A could bind to and transactivate the integrin α1 and α5 promoters. Treatment with the Rho inhibitor C3 transferase, the Rho-associated protein kinase (ROCK) inhibitor Y27632 or with shMRTF-A inhibited both the upregulation of α1, α5, and β1 as well as angiogenesis. Furthermore, in human umbilical vein endothelial cells (HUVECs), MRTF-A deletion led to marked reductions in cell migration and vessel network formation compared with the control. These data demonstrate that Rho/MRTF-A signaling is an important mediator that controls integrin gene expression during MSC-mediated angiogenic processes. Rui Zhang, Nan Wang, Man Zhang, Li-Nan Zhang, Zhi-Xia Guo, Xue-Gang Luo, Hao Zhou, Hong-Peng He, and Tong-Cun Zhang Copyright © 2015 Rui Zhang et al. All rights reserved. Differences in the Gene Expression Profiles of Slow- and Fast-Forming Preinduced Pluripotent Stem Cell Colonies Tue, 07 Apr 2015 10:12:04 +0000 Induced pluripotent stem cells (iPSCs) are generated through a gradual process in which somatic cells undergo a number of stochastic events. In this study, we examined whether two different doxycycline-inducible iPSCs, slow-forming 4F2A-iPSCs and fast-forming NGFP-iPSCs, have equivalent levels of pluripotency. Multiplex reverse-transcriptase PCR generated gene expression profiles (GEPs) of 13 pluripotency genes in single initially formed-iPSC (if-iPSC) colonies of NGFP and 4F2A group. Assessment of GEP difference using a weighted root mean square deviation (wRMSD) indicates that 4F2A if-iPSCs are more closely related to mESCs than NGFP if-iPSCs. Consistently, Nanog and Sox2 genes were more frequently derepressed in 4F2A if-iPSC group. We further examined 20 genes that are implicated in reprogramming. They were, overall, more highly expressed in NGFP if-iPSCs, differing from the pluripotency genes being more expressed in 4F2A if-iPSCs. wRMSD analysis for these reprogramming-related genes confirmed that the 4F2A if-iPSC colonies were less deviated from mESCs than the NGFP if-iPSC colonies. Our findings suggest that more important in attaining a better reprogramming is the mode of action by the given reprogramming factors, rather than the total activity of them exerting to the cells, as the thin-but-long-lasting mode of action in 4F2A if-iPSCs is shown to be more effective than its full-but-short-lasting mode in NGFP if-iPSCs. Sujin Kwon, Jung Sun Park, Byungkuk Min, and Yong-Kook Kang Copyright © 2015 Sujin Kwon et al. All rights reserved. Concise Review: Are Stimulated Somatic Cells Truly Reprogrammed into an ES/iPS-Like Pluripotent State? Better Understanding by Ischemia-Induced Multipotent Stem Cells in a Mouse Model of Cerebral Infarction Mon, 06 Apr 2015 11:45:54 +0000 Following the discovery of pluripotent stem (PS) cells such as embryonic stem (ES) and induced pluripotent stem (iPS) cells, there has been a great hope that injured tissues can be repaired by transplantation of ES/iPS-derived various specific types of cells such as neural stem cells (NSCs). Although PS cells can be induced by ectopic expression of Yamanaka’s factors, it is known that several stimuli such as ischemia/hypoxia can increase the stemness of somatic cells via reprogramming. This suggests that endogenous somatic cells acquire stemness during natural regenerative processes following injury. In this study, we describe whether somatic cells are converted into pluripotent stem cells by pathological stimuli without ectopic expression of reprogramming factors based on the findings of ischemia-induced multipotent stem cells in a mouse model of cerebral infarction. Takayuki Nakagomi, Akiko Nakano-Doi, Aya Narita, and Tomohiro Matsuyama Copyright © 2015 Takayuki Nakagomi et al. All rights reserved. Successful Isolation of Viable Adipose-Derived Stem Cells from Human Adipose Tissue Subject to Long-Term Cryopreservation: Positive Implications for Adult Stem Cell-Based Therapeutics in Patients of Advanced Age Sun, 05 Apr 2015 14:22:28 +0000 We examined cell isolation, viability, and growth in adipose-derived stem cells harvested from whole adipose tissue subject to different cryopreservation lengths (2–1159 days) from patients of varying ages (26–62 years). Subcutaneous abdominal adipose tissue was excised during abdominoplasties and was cryopreserved. The viability and number of adipose-derived stem cells isolated were measured after initial isolation and after 9, 18, and 28 days of growth. Data were analyzed with respect to cryopreservation duration and patient age. Significantly more viable cells were initially isolated from tissue cryopreserved <1 year than from tissue cryopreserved >2 years, irrespective of patient age. However, this difference did not persist with continued growth and there were no significant differences in cell viability or growth at subsequent time points with respect to cryopreservation duration or patient age. Mesenchymal stem cell markers were maintained in all cohorts tested throughout the duration of the study. Consequently, longer cryopreservation negatively impacts initial live adipose-derived stem cell isolation; however, this effect is neutralized with continued cell growth. Patient age does not significantly impact stem cell isolation, viability, or growth. Cryopreservation of adipose tissue is an effective long-term banking method for isolation of adipose-derived stem cells in patients of varying ages. Sean M. Devitt, Cynthia M. Carter, Raia Dierov, Scott Weiss, Robert P. Gersch, and Ivona Percec Copyright © 2015 Sean M. Devitt et al. All rights reserved. Dual Function of Wnt Signaling during Neuronal Differentiation of Mouse Embryonic Stem Cells Sun, 05 Apr 2015 12:16:22 +0000 Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/β-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells. To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induce β-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively (BIO, GSK3β inhibitor) or negatively (IWR-1-endo, Axin stabilizer) control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested that β-catenin/TCF1 complex directly regulated the expression of Sox1 during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation. Hanjun Kim, Sewoon Kim, Yonghee Song, Wantae Kim, Qi-Long Ying, and Eek-hoon Jho Copyright © 2015 Hanjun Kim et al. All rights reserved. Cell Cycle-Driven Heterogeneity: On the Road to Demystifying the Transitions between “Poised” and “Restricted” Pluripotent Cell States Sun, 05 Apr 2015 08:51:33 +0000 Cellular heterogeneity is now considered an inherent property of most stem cell types, including pluripotent stem cells, somatic stem cells, and cancer stem cells, and this heterogeneity can exist at the epigenetic, transcriptional, and posttranscriptional levels. Several studies have indicated that the stochastic activation of signaling networks may promote heterogeneity and further that this heterogeneity may be reduced by their inhibition. But why different cells in the same culture respond in a nonuniform manner to the identical exogenous signals has remained unclear. Recent studies now demonstrate that the cell cycle position directly influences lineage specification and specifically that pluripotent stem cells initiate their differentiation from the G1 phase. These studies suggest that cells in G1 are uniquely “poised” to undergo cell specification. G1 cells are therefore more prone to respond to differentiation cues, which may explain the heterogeneity of developmental factors, such as Gata6, and pluripotency factors, such as Nanog, in stem cell cultures. Overall, this raises the possibility that G1 serves as a “Differentiation Induction Point.” In this review, we will reexamine the literature describing heterogeneity of pluripotent stem cells, while highlighting the role of the cell cycle as a major determinant. Amar M. Singh Copyright © 2015 Amar M. Singh. All rights reserved. Reprogramming with Small Molecules instead of Exogenous Transcription Factors Wed, 01 Apr 2015 09:45:49 +0000 Induced pluripotent stem cells (iPSCs) could be employed in the creation of patient-specific stem cells, which could subsequently be used in various basic and clinical applications. However, current iPSC methodologies present significant hidden risks with respect to genetic mutations and abnormal expression which are a barrier in realizing the full potential of iPSCs. A chemical approach is thought to be a promising strategy for safety and efficiency of iPSC generation. Many small molecules have been identified that can be used in place of exogenous transcription factors and significantly improve iPSC reprogramming efficiency and quality. Recent studies have shown that the use of small molecules results in the generation of chemically induced pluripotent stem cells from mouse embryonic fibroblast cells. These studies might lead to new areas of stem cell research and medical applications, not only human iPSC by chemicals alone, but also safe generation of somatic stem cells for cell based clinical trials and other researches. In this paper, we have reviewed the recent advances in small molecule approaches for the generation of iPSCs. Tongxiang Lin and Shouhai Wu Copyright © 2015 Tongxiang Lin and Shouhai Wu. All rights reserved. Targeted Knockdown of RNA-Binding Protein TIAR for Promoting Self-Renewal and Attenuating Differentiation of Mouse Embryonic Stem Cells Tue, 31 Mar 2015 11:08:27 +0000 RNA-binding protein TIAR has been suggested to mediate the translational silencing of ARE-containing mRNAs. To analyze the functions of TIAR, we established RNAi and genetic rescue assays. We evaluated the expression of neuroectoderm markers Pax6 and nestin, mesoderm markers brachyury and Flk1, and hypoblast and definitive endoderm markers Sox17 and Gata6 during EB differentiation and found that knockdown TIAR expression restrained the differentiation of E14 cells. We assessed gene expression levels of Flk-1 and VE-cadherin and observed attenuated differentiation of E14 cells into endothelial cells upon downregulation of TIAR gene expression. As such, we hypothesized an essential role of TIAR related to EB differentiation. As TIAR inhibits the translation of c-myc, we proposed that downregulation of TIAR results in restrained differentiation of E14 cells, due in part to the function of c-myc. We found that TIAR inhibited c-myc expression at the translational level in E14 cells; accordingly, a reduction of TIAR expression promoted self-renewal of pluripotent cells and attenuated differentiation. Additionally, we established that TIAR inhibited TIA-1 expression at the translational level in E14 cells. Taken together, we have contributed to the understanding of the regulatory relationships between TIAR and both c-myc and TIA-1. Zhe Geng, Ping Li, Li Tan, and Houyan Song Copyright © 2015 Zhe Geng et al. All rights reserved. Adult Vascular Wall Resident Multipotent Vascular Stem Cells, Matrix Metalloproteinases, and Arterial Aneurysms Wed, 18 Mar 2015 06:16:32 +0000 Evidences have shown the presence of multipotent stem cells (SCs) at sites of arterial aneurysms: they can differentiate into smooth muscle cells (SMCs) and are activated after residing in a quiescent state in the vascular wall. Recent studies have implicated the role of matrix metalloproteinases in the pathogenesis of arterial aneurysms: in fact the increased synthesis of MMPs by arterial SMCs is thought to be a pivotal mechanism in aneurysm formation. The factors and signaling pathways involved in regulating wall resident SC recruitment, survival, proliferation, growth factor production, and differentiation may be also related to selective expression of different MMPs. This review explores the relationship between adult vascular wall resident multipotent vascular SCs, MMPs, and arterial aneurysms. Bruno Amato, Rita Compagna, Maurizio Amato, Raffaele Grande, Lucia Butrico, Alessio Rossi, Agostino Naso, Michele Ruggiero, Stefano de Franciscis, and Raffaele Serra Copyright © 2015 Bruno Amato et al. All rights reserved. Periodontal Ligament Stem Cells: Current Status, Concerns, and Future Prospects Mon, 16 Mar 2015 16:25:51 +0000 Periodontal ligament stem cells (PDLSCs), which reside in the perivascular space of the periodontium, possess characteristics of mesenchymal stem cells and are a promising tool for periodontal regeneration. Recently, great progress has been made in PDLSC transplantation. Investigators are attempting to maximize the proliferation and differentiation potential of PDLSCs by modifying culture conditions and applying growth factors. Nevertheless, problems remain. First, incomparability among different studies must be minimized by establishing standard guidelines for culture and identification of PDLSCs. Notably, attention should be paid to the biological safety of PDLSC transplantation. The present review updates the latest findings regarding PDLSCs and discusses standard criteria for culture and identification of PDLSCs. Finally, the review calls for careful consideration of PDLSC transplantation safety. Wenjun Zhu and Min Liang Copyright © 2015 Wenjun Zhu and Min Liang. All rights reserved. Development of a Xeno-Free Substrate for Human Embryonic Stem Cell Growth Mon, 16 Mar 2015 12:44:15 +0000 Traditionally, human embryonic stem cells (hESCs) are cultured on inactivated live feeder cells. For clinical application using hESCs, there is a requirement to minimize the risk of contamination with animal components. Extracellular matrix (ECM) derived from feeder cells is the most natural way to provide xeno-free substrates for hESC growth. In this study, we optimized the step-by-step procedure for ECM processing to develop a xeno-free ECM that supports the growth of undifferentiated hESCs. In addition, this newly developed xeno-free substrate can be stored at 4°C and is ready to use upon request, which serves as an easier way to amplify hESCs for clinical applications. Hailin Zhu, Jinliang Yang, Yuquan Wei, and Harry Huimin Chen Copyright © 2015 Hailin Zhu et al. All rights reserved. Beneficial Effects of Coculturing Synovial Derived Mesenchymal Stem Cells with Meniscus Fibrochondrocytes Are Mediated by Fibroblast Growth Factor 1: Increased Proliferation and Collagen Synthesis Mon, 16 Mar 2015 09:33:33 +0000 Meniscus reconstruction is in great need for orthopedic surgeons. Meniscal fibrochondrocytes transplantation was proposed to regenerate functional meniscus, with limited donor supply. We hypothesized that coculture of synovial mesenchymal stem cells (SSC) with meniscal fibrochondrocytes (me-CH) can support matrix production of me-CH, thus reducing the number of me-CH needed for meniscus reconstruction. A pellet coculture system of human SSC and me-CH was used in this study. Enhanced glycosaminoglycans (GAG) in coculture pellets were demonstrated by Alcian blue staining and GAG quantification, when compared to monoculture. More collagen synthesis was shown in coculture pellets by hydroxyproline assay. Increased proliferation of me-CH was observed in coculture. Data from BrdU staining and ELISA demonstrated that conditioned medium of SSCs enhanced the proliferation and collagen synthesis of me-CH, and this effect was blocked by neutralizing antibody against fibroblast growth factor 1 (FGF1). Western blot showed that conditioned medium of SSCs can activate mitogen-activated protein kinase (MAPK) signaling pathways by increasing the phosphorylation of mitogen-activated regulated protein kinase 1/2 (MEK) and extracellular-signal-regulated kinases 1/2 (ERK). Overall, this study provided evidence that synovial MSCs can support proliferation and collagen synthesis of fibrochondrocytes, by secreting FGF1. Coimplantation of SSC and me-CH could be a useful strategy for reconstructing meniscus. Xuanhe Song, Yaoping Xie, Yang Liu, Ming Shao, and Wenbo Wang Copyright © 2015 Xuanhe Song et al. All rights reserved. The Chondrogenic Induction Potential for Bone Marrow-Derived Stem Cells between Autologous Platelet-Rich Plasma and Common Chondrogenic Induction Agents: A Preliminary Comparative Study Mon, 16 Mar 2015 09:14:09 +0000 The interests in platelet-rich plasma (PRP) and their application in stem cell therapy have contributed to a better understanding of the basic biology of the prochondrogenesis effect on bone marrow-derived stem cells (BMSCs). We aimed at comparing the effect of autologous PRP with common chondrogenic induction agents (CCIAs) on the chondrogenic differentiation of BMSCs. Rabbit BMSCs were isolated and characterized by flow cytometry and differentiated towards adipocytes and osteoblasts. The chondrogenic response of BMSCs to autologous PRP and CCIAs which included transforming growth factor-β1 (TGF-β1), dexamethasone (DEX), and vitamin C (Vc) was examined by cell pellet culture. The isolated BMSCs after two passages highly expressed CD29 and CD44 but minimally expressed CD45. The osteogenic and adipogenic differentiation potentials of the isolated BMSCs were also confirmed. Compared with common CCIAs, autologous PRP significantly upregulated the chondrogenic related gene expression, including Col-2, AGC, and Sox-9. Osteogenic related gene expression, including Col-1 and OCN, was not of statistical significance between these two groups. Thus, our data shows that, compared with common chondrogenic induction agents, autologous PRP can be more effective in promoting the chondrogenesis of BMSCs. Shan-zheng Wang, Qing Chang, Xiang-fei Kong, and Chen Wang Copyright © 2015 Shan-zheng Wang et al. All rights reserved. Bone Marrow-Derived, Neural-Like Cells Have the Characteristics of Neurons to Protect the Peripheral Nerve in Microenvironment Mon, 16 Mar 2015 06:17:34 +0000 Effective repair of peripheral nerve defects is difficult because of the slow growth of new axonal growth. We propose that “neural-like cells” may be useful for the protection of peripheral nerve destructions. Such cells should prolong the time for the disintegration of spinal nerves, reduce lesions, and improve recovery. But the mechanism of neural-like cells in the peripheral nerve is still unclear. In this study, bone marrow-derived neural-like cells were used as seed cells. The cells were injected into the distal end of severed rabbit peripheral nerves that were no longer integrated with the central nervous system. Electromyography (EMG), immunohistochemistry, and transmission electron microscopy (TEM) were employed to analyze the development of the cells in the peripheral nerve environment. The CMAP amplitude appeared during the 5th week following surgery, at which time morphological characteristics of myelinated nerve fiber formation were observed. Bone marrow-derived neural-like cells could protect the disintegration and destruction of the injured peripheral nerve. Shi-lei Guo, Zhi-ying Zhang, Yan Xu, Yun-xia Zhi, Chang-jie Han, Yu-hao Zhou, Fang Liu, Hai-yan Lin, and Chuan-sen Zhang Copyright © 2015 Shi-lei Guo et al. All rights reserved. Sonic Hedgehog Produced by Bone Marrow-Derived Mesenchymal Stromal Cells Supports Cell Survival in Myelodysplastic Syndrome Sun, 15 Mar 2015 08:38:26 +0000 The role of marrow microenvironment in the pathogenesis of myelodysplastic syndrome (MDS) remains controversial. Therefore, we studied the influence of bone marrow-derived mesenchymal stromal cells (BMSCs) from patients with different risk types of MDS on the survival of the MDS cell lines SKM-1 and MUTZ-1. We first demonstrated that the expression of Sonic hedgehog (Shh), smoothened (Smo), and glioma-associated oncogene homolog 1 (Gli1) was increased in MDS patients ; the increase in expression was positively correlated with the presence of high-risk factors. The Shh signaling inhibitor, cyclopamine, inhibited high-risk MDS BMSC-induced survival of SKM-1 and MUTZ-1 cells, suggesting a role for Shh signaling in MDS cell survival. Furthermore, cyclopamine-mediated inhibition of Shh signaling in SKM-1 and MUTZ-1 cells resulted in decreased DNMT1 expression and cell survival; however, exogenous Shh peptide had the opposite effect, suggesting that Shh signaling could regulate the expression of DNMT1, thereby modulating cell survival in MDS. In addition, the apoptosis of SKM-1 and MUTZ-1 cell increased significantly when cultured with cyclopamine and a demethylation agent, 5-Aza-2′-deoxycytidine. These findings suggest that Shh signaling from BMSCs is important in the pathogenesis of MDS and could play a role in disease progression by modulating methylation. Jixue Zou, Yan Hong, Yin Tong, Ju Wei, Youwen Qin, Shan Shao, Chun Wang, and Kun Zhou Copyright © 2015 Jixue Zou et al. All rights reserved. Intravenous Administration of Bone Marrow-Derived Mesenchymal Stem Cells Induces a Switch from Classical to Atypical Symptoms in Experimental Autoimmune Encephalomyelitis Mon, 09 Mar 2015 11:55:49 +0000 Potent immunosuppressive and regenerative properties of mesenchymal stem cells (MSCs) position them as a novel therapy for autoimmune diseases. This research examines the therapeutic effect of MSCs administration at different disease stages in experimental autoimmune encephalomyelitis (EAE). Classical and atypical scores of EAE, associated with Th1 and Th17 response, respectively, and also Treg lymphocytes, were evaluated. MSCs administration at the onset (EAE+MSConset) induced an important amelioration of the clinical signs and less lasting effect at the peak of EAE (EAE+MSCpeak). No effect was observed when MSCs were applied after EAE stabilization (EAE+MSClate). Surprisingly, EAE atypical signs were detected in EAE+MSCpeak and EAE+MSClate mice. However, no correlation was found in Th17/Th1 ratio. Interestingly, regardless of time administration, MSCs significantly reduced IL-6 and also T-bet, RORγT, and Foxp3 mRNA levels in brain samples of EAE mice. The downregulation of IL-6 could restore the well-functioning of the blood-brain barrier of EAE mice, correlated with a decreased number of brain infiltrating leukocytes. These results suggest that the inflammatory status is important to be considered for administering MSCs in autoimmune pathologies, leading to a further research to clarify the effect of MSCs for multiple sclerosis. Mónica Kurte, Javiera Bravo-Alegría, Alexander Torres, Vania Carrasco, Cristina Ibáñez, Ana María Vega-Letter, Catalina Fernández-O’Ryan, Carlos E. Irarrázabal, Fernando E. Figueroa, Rodrigo A. Fuentealba, Claudia Riedel, and Flavio Carrión Copyright © 2015 Mónica Kurte et al. All rights reserved. Comparative Investigation of Human Amniotic Epithelial Cells and Mesenchymal Stem Cells for Application in Bone Tissue Engineering Thu, 05 Mar 2015 09:13:59 +0000 Emerging evidence suggests amniotic epithelial cells (AECs) as a promising source of progenitor cells in regenerative medicine and bone tissue engineering. However, investigations comparing the regenerative properties of AECs with other sources of stem cells are particularly needed before the feasibility of AECs in bone tissue engineering can be determined. This study aimed to compare human amniotic epithelial cells (hAECs), human bone marrow mesenchymal stem cells (hBMSCs), and human amniotic fluid derived mesenchymal stem cells (hAFMSCs) in terms of their morphology, proliferation, immunophenotype profile, and osteogenic capacity in vitro and in vivo. Not only greatly distinguished by cell morphology and proliferation, hAECs, hAFMSCs, and hBMSCs exhibited remarkably different signature regarding immunophenotypical profile. Microarray analysis revealed a different expression profile of genes involved in ossification along the three cell sources, highlighting the impact of different anatomical origin and molecular response to osteogenic induction on the final tissue-forming potential. Furthermore, our data indicated a potential role of FOXC2 in early osteogenic commitment. Jiawen Si, Jiewen Dai, Jianjun Zhang, Sha Liu, Jing Gu, Jun Shi, Steve G. F. Shen, and Lihe Guo Copyright © 2015 Jiawen Si et al. All rights reserved. Comparisons of Mouse Mesenchymal Stem Cells in Primary Adherent Culture of Compact Bone Fragments and Whole Bone Marrow Tue, 03 Mar 2015 13:44:53 +0000 The purification of mouse bone marrow mesenchymal stem cells (BMSCs) by using the standard method of whole bone marrow adherence to plastic still remains ineffective. An increasing number of studies have indicated compact bone as an alternative source of BMSCs. We isolated BMSCs from cultured compact bone fragments and investigated the proliferative capacity, surface immunophenotypes, and osteogenic and adipogenic differentiations of the cells after the first trypsinization. The fragment culture was based on the fact that BMSCs were assembled in compact bones. Thus, the procedure included flushing bone marrow out of bone cavity and culturing the fragments without any collagenase digestion. The cell yield from cultured fragments was slightly less than that from cultured bone marrow using the same bone quantity. However, the trypsinized cells from cultured fragments exhibited significantly higher proliferation and were accompanied with more CD90 and CD44 expressions and less CD45 expression. The osteogenic and adipogenic differentiation capacity of cells from cultured fragments were better than those of cells from bone marrow. The directly adherent culture of compact bone is suitable for mouse BMSC isolation, and more BMSCs with potentially improved proliferation capacity can be obtained in the primary culture. Yiting Cai, Tianshu Liu, Fang Fang, Chengliang Xiong, and Shiliang Shen Copyright © 2015 Yiting Cai et al. All rights reserved. Mesenchymal Stem Cell Therapy in Nonhematopoietic Diseases Sun, 01 Mar 2015 07:52:26 +0000 Katherine Athayde Teixeira de Carvalho, Gustav Steinhoff, and Juan Carlos Chachques Copyright © 2015 Katherine Athayde Teixeira de Carvalho et al. All rights reserved. Bone Marrow-Derived Mesenchymal Stem Cells Repair Necrotic Pancreatic Tissue and Promote Angiogenesis by Secreting Cellular Growth Factors Involved in the SDF-1α/CXCR4 Axis in Rats Wed, 25 Feb 2015 08:24:19 +0000 Acute pancreatitis (AP), a common acute abdominal disease, 10%–20% of which can evolve into severe acute pancreatitis (SAP), is of significant morbidity and mortality. Bone marrow-derived mesenchymal stem cells (BMSCs) have been reported to have a potential therapeutic role on SAP, but the specific mechanism is unclear. Therefore, we conducted this experiment to shed light on the probable mechanism. We validated that SDF-1α significantly stimulated the expressions of VEGF, ANG-1, HGF, TGF-β, and CXCR4 in BMSCs, which were inhibited by its receptor agonist, AMD3100. The capacities of proliferation, migration, and repair of human umbilical vein endothelial cells were enhanced by BMSCs supernatant. Meanwhile, BMSCs supernatant could also promote angiogenesis, especially after the stimulation with SDF-1α. In vivo, the migration of BMSCs was regulated by SDF-1α/CXCR4 axis. Moreover, transplanted BMSCs could significantly alleviate SAP, reduce the systematic inflammation (TNF-, IL- IL-6↓, IL-4↑, IL-10↑, and TGF-, and promote tissue repair and angiogenesis (VEGF↑, ANG-1↑, HGF↑, TGF-, and CD31↑), compared with the SAP and anti-CXCR4 groups. Taken together, the results showed that BMSCs ameliorated SAP and the SDF-1α/CXCR4 axis was involved in the repair and regeneration process. Daohai Qian, Jian Gong, Zhigang He, Jie Hua, Shengping Lin, Chenglei Xu, Hongbo Meng, and Zhenshun Song Copyright © 2015 Daohai Qian et al. All rights reserved. Diversity of Epithelial Stem Cell Types in Adult Lung Tue, 24 Feb 2015 13:02:53 +0000 Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. Feng Li, Jinxi He, Jun Wei, William C. Cho, and Xiaoming Liu Copyright © 2015 Feng Li et al. All rights reserved. Neuroprotective and Antiapoptotic Activity of Lineage-Negative Bone Marrow Cells after Intravitreal Injection in a Mouse Model of Acute Retinal Injury Tue, 24 Feb 2015 11:28:49 +0000 We investigated effects of bone marrow-derived, lineage-negative cell (Lin−BMC) transplantation in acute retinal injury. Lin−BMCs were intravitreally injected into murine eyes at 24 h after NaIO3-induced injury. Morphology, function, and expression of apoptosis-related genes, including brain-derived neurotrophic factor (BDNF) and its receptor, were assessed in retinas at 7 days, 28 days, and 3 months after transplantation. Moreover, global gene expression at day 7 was analyzed by RNA arrays. We observed that Lin−BMCs integrated into outer retinal layers improving morphological retinal structure and induced molecular changes such as downregulation of proapoptotic caspase-3 gene, a decrease in BAX/BCL-2 gene ratio, and significant elevation of BDNF expression. Furthermore, transplanted Lin−BMCs differentiated locally into cells with a macrophage-like phenotype. Finally, Lin−BMCs treatment was associated with generation of two distinct transcriptomic patterns. The first relates to downregulated genes associated with regulation of neuron cell death and apoptosis, response to oxidative stress/hypoxia and external stimuli, and negative regulation of cell proliferation. The second relates to upregulated genes associated with neurological system processes and sensory perception. Collectively, our data demonstrate that transplanted Lin−BMCs exert neuroprotective function against acute retinal injury and this effect may be associated with their antiapoptotic properties and ability to express neurotrophic factors. Anna Machalińska, Dorota Rogińska, Ewa Pius-Sadowska, Miłosz P. Kawa, Edyta Paczkowska, Michał Rudnicki, Renata Lejkowska, Bartłomiej Baumert, Barbara Wiszniewska, and Bogusław Machaliński Copyright © 2015 Anna Machalińska et al. All rights reserved. A Clinical Study of Autologous Bone Marrow Mononuclear Cells for Cerebral Palsy Patients: A New Frontier Wed, 18 Feb 2015 13:43:07 +0000 Cerebral palsy is a nonprogressive heterogeneous group of neurological disorders with a growing rate of prevalence. Recently, cellular therapy is emerging as a potential novel treatment strategy for cerebral palsy. The various mechanisms by which cellular therapy works include neuroprotection, immunomodulation, neurorestoration, and neurogenesis. We conducted an open label, nonrandomized study on 40 cases of cerebral palsy with an aim of evaluating the benefit of cellular therapy in combination with rehabilitation. These cases were administered autologous bone marrow mononuclear cells intrathecally. The follow-up was carried out at 1 week, 3 months, and 6 months after the intervention. Adverse events of the treatment were also monitored in this duration. Overall, at six months, 95% of patients showed improvements. The study population was further divided into diplegic, quadriplegic, and miscellaneous group of cerebral palsy. On statistical analysis, a significant association was established between the symptomatic improvements and cell therapy in diplegic and quadriplegic cerebral palsy. PET-CT scan done in 6 patients showed metabolic improvements in areas of the brain correlating to clinical improvements. The results of this study demonstrate that cellular therapy may accelerate the development, reduce disability, and improve the quality of life of patients with cerebral palsy. Alok Sharma, Hemangi Sane, Nandini Gokulchandran, Pooja Kulkarni, Sushant Gandhi, Jyothi Sundaram, Amruta Paranjape, Akshata Shetty, Khushboo Bhagwanani, Hema Biju, and Prerna Badhe Copyright © 2015 Alok Sharma et al. All rights reserved. Effects of Transient Hypoxia versus Prolonged Hypoxia on Satellite Cell Proliferation and Differentiation In Vivo Wed, 18 Feb 2015 13:01:45 +0000 The microenvironment of the injury site can have profound effects on wound healing. Muscle injury results in ischemia leading to short-term local hypoxia, but there are conflicting reports on the role of hypoxia on the myogenic program in vivo and in vitro. In our rat model of mitochondrial restoration (MR), temporary upregulation of mitochondrial activity by a cocktail of organelle-encoded RNAs results in satellite cell proliferation and initiation of myogenesis. We now report that MR leads to a transient hypoxic response in situ. Inhibition of hypoxia by lowering mitochondrial O2 consumption, either by respiratory electron transport inhibitors, or by NO-mediated inhibition of O2 binding to cytochrome c oxidase, resulted in exacerbation of inflammation. Lentivirus-mediated knockdown of hypoxia-inducible factor 1α (HIF1α) or of Notch signaling components had a similar effect, and pharmacologic inhibition of HIF or Notch reduced the number of proliferating Pax7+ cells. In contrast, a prolonged hypoxic response induced either by uncoupling of respiration from oxidative phosphorylation or through HIF stabilization by dimethyloxalylglycine (DMOG) had an immediate anti-inflammatory effect. Although significant satellite cell proliferation occurred in presence of DMOG, expression of differentiation markers was affected. These results emphasize the importance of transient hypoxia as opposed to prolonged hypoxia for myogenesis. Sukanta Jash and Samit Adhya Copyright © 2015 Sukanta Jash and Samit Adhya. All rights reserved. Immune Suppressive Effects of Tonsil-Derived Mesenchymal Stem Cells on Mouse Bone-Marrow-Derived Dendritic Cells Mon, 16 Feb 2015 14:00:25 +0000 Mesenchymal stem cells (MSCs) are considered valuable sources for cell therapy because of their immune regulatory function. Here, we investigated the effects of tonsil-derived MSCs (T-MSCs) on the differentiation, maturation, and function of dendritic cells (DCs). We examined the effect of T-MSCs on differentiation and maturation of bone-marrow- (BM-) derived monocytes into DCs and we found suppressive effect of T-MSCs on DCs via direct contact as well as soluble mediators. Moreover, T cell proliferation, normally increased in the presence of DCs, was inhibited by T-MSCs. Differentiation of CD4+ T cell subsets by the DC-T cell interaction also was inhibited by T-MSCs. The soluble mediators suppressed by T-MSCs were granulocyte-macrophage colony-stimulating factor (GM-CSF), RANTES, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Taken together, T-MSCs exert immune modulatory function via suppression of the differentiation, maturation, and function of BM-derived DCs. Our data suggests that T-MSCs could be used as a novel source of stem cell therapy as immune modulators. Minhwa Park, Yu-Hee Kim, Jung-Hwa Ryu, So-Youn Woo, and Kyung-Ha Ryu Copyright © 2015 Minhwa Park et al. All rights reserved. CXCL13 Promotes Osteogenic Differentiation of Mesenchymal Stem Cells by Inhibiting miR-23a Expression Mon, 16 Feb 2015 13:33:12 +0000 CXC chemokines are essential for osteogenic differentiation of bone mesenchymal stem cells (BMSCs) for use in bone tissue engineering and regenerative medicine in clinical settings. However, an accurate understanding of the underlying mechanisms is still needed. In this study, we analyzed the effects of CXC chemokine ligand-13 (CXCL13) on osteogenic differentiation of rat BMSCs and initiated a preliminary discussion on possible mechanisms. BMSCs were isolated from bone marrow of rat and incubated with CXCL13 recombinant protein in differentiation medium. The main osteogenesis indexes were alkaline phosphatase (ALP) activity and calcium nodes. Expression of Runx2 and CXCR5 was determined using western blot, while miRNAs were determined with quantitative-RT-PCR. Si-CXCR5 was transfected into MSCs to silence CXCR5. A miRNA-23a mimic was transfected into BMSCs for overexpression of miRNA-23a. Recombinant CXCL13 induced ALP activity, deposition of calcium salts, and formation of calcium nodes, and it also increased expression of Runx2. The expression of recombinant CXCL13 suppressed expression of miRNA-23a. Overexpression of miR-23a reversed CXCL13 induced-osteogenic differentiation of BMSCs and expression of Runx2. Recombinant CXCL13 attenuated the interaction of miRNA-23a with the Runx2 3′UTR. Silencing of CXCR5 abrogated recombinant CXCL13-induced downregulation of miRNA-23a expression. In summary, CXCL13 promotes osteogenic differentiation of BMSCs by inhibiting miR-23a expression. Feng Tian, Xiang-lu Ji, Wan-an Xiao, Bin Wang, and Fei Wang Copyright © 2015 Feng Tian et al. All rights reserved. Matrix Metalloproteinase 9 Secreted by Hypoxia Cardiac Fibroblasts Triggers Cardiac Stem Cell Migration In Vitro Thu, 12 Feb 2015 07:03:04 +0000 Cessation of blood supply due to myocardial infarction (MI) leads to complicated pathological alteration in the affected regions. Cardiac stem cells (CSCs) migration plays a major role in promoting recovery of cardiac function and protecting cardiomyocytes in post-MI remodeling. Despite being the most abundant cell type in the mammalian heart, cardiac fibroblasts (CFs) were underestimated in the mechanism of CSCs migration. Our objective in this study is therefore to investigate the migration related factors secreted by hypoxia CFs in vitro and the degree that they contribute to CSCs migration. We found that supernatant from hypoxia induced CFs could accelerate CSCs migration. Four migration-related cytokines were reported upregulated both in mRNA and protein levels. Upon adding antagonists of these cytokines, the number of migration cells significantly declined. When the cocktail antagonists of all above four cytokines were added, the migration cells number reduced to the minimum level. Besides, MMP-9 had an important effect on triggering CSCs migration. As shown in our results, MMP-9 induced CSCs migration and the underlying mechanism might involve TNF-α signaling which induced VEGF and MMP-9 expression. Qing Gao, Maojuan Guo, Wenyun Zeng, Yijing Wang, Lin Yang, Xiaoli Pang, Huhu Li, Yanrong Suo, Xijuan Jiang, and Chunquan Yu Copyright © 2015 Qing Gao et al. All rights reserved. Alkaline Phosphatase in Stem Cells Thu, 12 Feb 2015 06:37:39 +0000 Alkaline phosphatase is an enzyme commonly expressed in almost all living organisms. In humans and other mammals, determinations of the expression and activity of alkaline phosphatase have frequently been used for cell determination in developmental studies and/or within clinical trials. Alkaline phosphatase also seems to be one of the key markers in the identification of pluripotent embryonic stem as well as related cells. However, alkaline phosphatases exist in some isoenzymes and isoforms, which have tissue specific expressions and functions. Here, the role of alkaline phosphatase as a stem cell marker is discussed in detail. First, we briefly summarize contemporary knowledge of mammalian alkaline phosphatases in general. Second, we focus on the known facts of its role in and potential significance for the identification of stem cells. Kateřina Štefková, Jiřina Procházková, and Jiří Pacherník Copyright © 2015 Kateřina Štefková et al. All rights reserved.