Characterization and Evaluation of Neuronal Trans-Differentiation with Electrophysiological Properties of Mesenchymal Stem Cells Isolated from Porcine Endometrium

Baregundi Subbarao, Raghavendra,Ullah, Imran,Kim, Eun-Jin,Jang, Si-Jung,Lee, Won-Jae,Jeon, Ryoung Hoon,Kang, Dawon,Lee, Sung-Lim,Park, Bong-Wook,Rho, Gyu-Jin MDPI 2015 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.16 No.5

<P>Endometrial stromal cells (EMSCs) obtained from porcine uterus (<I>n</I> = 6) were positive for mesenchymal stem cell markers (CD29, CD44 and CD90), and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, <I>CD105</I>, <I>CD140b</I>, and <I>CD144</I> by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipocytes and osteocytes was confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Adipocyte (<I>FABP</I>, <I>LPL</I>, <I>AP2</I>) and osteocyte specific genes (<I>ON</I>, <I>BG</I>, <I>RUNX2</I>) in differentiated EMSCs showed significant (<I>p</I> < 0.05) increase in expression compared to undifferentiated control cells. Neurogenic transdifferentiation of EMSCs exhibited distinctive dendritic morphology with axon projections and neuronal specific genes, <I>NFM</I>, <I>NGF</I>, <I>MBP</I>, <I>NES</I>, <I>B3T</I> and <I>MAP2</I> and proteins, B3T, NFM, NGF, and TRKA were positively expressed in neuronal differentiated cells. Functional analysis of neuronal differentiated EMSCs displayed voltage-dependence and kinetics for transient outward K<SUP>+</SUP> currents (<I>I</I><SUB>to</SUB>), at holding potential of −80 mV, Na<SUP>+</SUP> currents and during current clamp, neuronal differentiated EMSCs was more negative than that of control EMSCs. Porcine EMSCs is a suitable model for studying molecular mechanism of transdifferentiation, assessment of electrophysiological properties and their efficiency during <I>in vivo</I> transplantation.</P>

Human mesenchymal stem cells - current trends and future prospective

Ullah, Imran,Subbarao, Raghavendra ,Baregundi,Rho, Gyu ,Jin Portland Press Ltd. 2015 Bioscience reports Vol.35 No.2

<▼1><P>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 <I>in vitro</I> culturing, <I>in vitro</I> differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.</P></▼1><▼2><P>In this review, we highlighted recent research findings in the area of human mesenchymal stem cells, its application in the treatment of chronic diseases and its use in human clinical trials.</P></▼2>

Differentiation potential of mesenchymal stem cells isolated from human dental tissues into nonmesodermal lineage

전병균,장시정,박지성,Raghavendra Baregundi Subbarao,정계준,박봉욱,노규진 한국통합생물학회 2015 Animal cells and systems Vol.19 No.5

Mesenchymal stem cells (MSCs) possess the ability to differentiate into non-mesodermal lineage, and examining their multipotency will be beneficial for application in regenerative medicine. The present study investigated the differentiation capacity into neuronal cells of ectodermal lineage and pancreatic cells of endodermal lineage in each of MSC lines isolated from three samples of human dental papilla tissues (DPSCs). Isolated DPSC lines expressed CD13, CD44, CD73, CD90 and CD105 cell surface markers, and OCT-4, NANOG and SOX-2 transcription factors. Further, all DPSC lines differentiated into osteocytes, adipocytes and chondrocytes of mesodermal lineage, whereas telomerase activity was at a low level in all isolated DPSC lines. Following induction into neuronal cells of ectodermal lineage, the neuron-like morphological alterations and expression of neuro-filament M by immunocytochemical staining was observed in all types of DPSCs, and expression of neuronal cell-specific transcripts, NSE, MAP-2, and NESTIN was further confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Moreover, following induction into pancreatic cells of endodermal lineage, all DPSC lines exhibited morphological alterations with DTZ-positive spheroid clusters, and expression of pancreatic cell-specific transcripts, INSULIN, PDX-1, and GLUT-2, was positively detected by RT-PCR. However, some of these clusters were negatively reacted with DTZ staining. The present results demonstrated that DPSCs exhibit differentiation capacity into neuronal and pancreatic cells of non-mesodermal lineage, and DPSCs could be an alternative source of MSCs for clinical applications.

Comparison of Immunomodulation Properties of Porcine Mesenchymal Stromal/Stem Cells Derived from the Bone Marrow, Adipose Tissue, and Dermal Skin Tissue

Ock, Sun-A,Baregundi Subbarao, Raghavendra,Lee, Yeon-Mi,Lee, Jeong-Hyeon,Jeon, Ryoung-Hoon,Lee, Sung-Lim,Park, Ji Kwon,Hwang, Sun-Chul,Rho, Gyu-Jin Hindawi Publishing Corporation 2016 Stem cells international Vol.2016 No.-

<P>Mesenchymal stromal/stem cells (MSCs) demonstrate immunomodulation capacity that has been implicated in the reduction of graft-versus-host disease. Accordingly, we herein investigated the capacity of MSCs derived from several tissue sources to modulate both proinflammatory (interferon [IFN] <I>γ</I> and tumor necrosis factor [TNF] <I>α</I>) and immunosuppressive cytokines (transforming growth factor [TGF] <I>β</I> and interleukin [IL] 10) employing xenogeneic human MSC-mixed lymphocyte reaction (MLR) test. Bone marrow-derived MSCs showed higher self-renewal capacity with relatively slow proliferation rate in contrast to adipose-derived MSCs which displayed higher proliferation rate. Except for the lipoprotein gene, there were no marked changes in osteogenesis- and adipogenesis-related genes following in vitro differentiation; however, the histological marker analysis revealed that adipose MSCs could be differentiated into both adipose and bone tissue. TGF<I>β</I> and IL10 were detected in adipose MSCs and bone marrow MSCs, respectively. However, skin-derived MSCs expressed both IFN<I>γ</I> and IL10, which may render them sensitive to immunomodulation. The xenogeneic human MLR test revealed that MSCs had a partial immunomodulation capacity, as proliferation of activated and resting peripheral blood mononuclear cells was not affected, but this did not differ among MSC sources. MSCs were not tumorigenic when introduced into immunodeficient mice. We concluded that the characteristics of MSCs are tissue source-dependent and their in vivo application requires more in-depth investigation regarding their precise immunomodulation capacities.</P>

DMSO‐ and Serum‐Free Cryopreservation of Wharton's Jelly Tissue Isolated From Human Umbilical Cord

Shivakumar, Sharath Belame,Bharti, Dinesh,Subbarao, Raghavendra Baregundi,Jang, Si‐,Jung,Park, Ji‐,Sung,Ullah, Imran,Park, Ji‐,Kwon,Byun, June‐,Ho,Park, Bong‐,Wook,Rho, G John Wiley and Sons Inc. 2016 Journal of cellular biochemistry Vol.117 No.10

<P><B>ABSTRACT</B></P><P>The facile nature of mesenchymal stem cell (MSC) acquisition in relatively large numbers has made Wharton's jelly (WJ) tissue an alternative source of MSCs for regenerative medicine. However, freezing of such tissue using dimethyl sulfoxide (DMSO) for future use impedes its clinical utility. In this study, we compared the effect of two different cryoprotectants (DMSO and cocktail solution) on post‐thaw cell behavior upon freezing of WJ tissue following two different freezing protocols (Conventional [−1°C/min] and programmed). The programmed method showed higher cell survival rate compared to conventional method of freezing. Further, cocktail solution showed better cryoprotection than DMSO. Post‐thaw growth characteristics and stem cell behavior of Wharton's jelly mesenchymal stem cells (WJMSCs) from WJ tissue cryopreserved with a cocktail solution in conjunction with programmed method (Prog‐Cock) were comparable with WJMSCs from fresh WJ tissue. They preserved their expression of surface markers, pluripotent factors, and successfully differentiated in vitro into osteocytes, adipocytes, chondrocytes, and hepatocytes. They also produced lesser annexin‐V‐positive cells compared to cells from WJ tissue stored using cocktail solution in conjunction with the conventional method (Conv‐Cock). Real‐time PCR and Western blot analysis of post‐thaw WJMSCs from Conv‐Cock group showed significantly increased expression of pro‐apoptotic factors (BAX, p53, and p21) and reduced expression of anti‐apoptotic factor (BCL2) compared to WJMSCs from the fresh and Prog‐Cock group. Therefore, we conclude that freezing of fresh WJ tissue using cocktail solution in conjunction with programmed freezing method allows for an efficient WJ tissue banking for future MSC‐based regenerative therapies. J. Cell. Biochem. 117: 2397–2412, 2016. © 2016 The Authors. <I>Journal of Cellular Biochemistry</I> published by Wiley Periodicals, Inc.</P>

Research Advancements in Porcine Derived Mesenchymal Stem Cells

Bharti, Dinesh,Shivakumar, Sharath Belame,Subbarao, Raghavendra Baregundi,Rho, Gyu-Jin Bentham Science Publishers 2016 Current stem cell research & therapy Vol.11 No.1

<P>In the present era of stem cell biology, various animals such as Mouse, Bovine, Rabbit and Porcine have been tested for the efficiency of their mesenchymal stem cells (MSCs) before their actual use for stem cell based application in humans. Among them pigs have many similarities to humans in the form of organ size, physiology and their functioning, therefore they have been considered as a valuable model system for <I>in vitro</I> studies and preclinical assessments. Easy assessability, few ethical issues, successful MSC isolation from different origins like bone marrow, skin, umbilical cord blood, Wharton’s jelly, endometrium, amniotic fluid and peripheral blood make porcine a good model for stem cell therapy. Porcine derived MSCs (pMSCs) have shown greater <I>in vitro</I> differentiation and transdifferention potential towards mesenchymal lineages and specialized lineages such as cardiomyocytes, neurons, hepatocytes and pancreatic beta cells. Immunomodulatory and low immunogenic profiles as shown by autologous and heterologous MSCs proves them safe and appropriate models for xenotransplantation purposes. Furthermore, tissue engineered stem cell constructs can be of immense importance in relation to various osteochondral defects which are difficult to treat otherwise. Using pMSCs successful treatment of various disorders like Parkinson’s disease, cardiac ischemia, hepatic failure, has been reported by many studies. Here, in this review we highlight current research findings in the area of porcine mesenchymal stem cells dealing with their isolation methods, differentiation ability, transplantation applications and their therapeutic potential towards various diseases.</P>

Comparative analysis of human Wharton’s jelly mesenchymal stem cells derived from different parts of the same umbilical cord

Bharti, Dinesh,Shivakumar, Sharath Belame,Park, Ji-Kwon,Ullah, Imran,Subbarao, Raghavendra Baregundi,Park, Ji-Sung,Lee, Sung-Lim,Park, Bong-Wook,Rho, Gyu-Jin Springer Berlin Heidelberg 2018 Cell and tissue research Vol.372 No.1

<P>Easy isolation, lack of ethical issues, high proliferation, multi-lineage differentiation potential and immunomodulatory properties of umbilical cord (UC)-derived mesenchymal stem cells (MSCs) make them a valuable tool in stem cell research. Recently, Wharton’s jelly (WJ) was proven as the best MSC source among various compartments of UC. However, it is still unclear whether or not Wharton’s jelly-derived MSCs (WJMSCs) from different parts of the whole cord exhibit the same characteristics. There may be varied MSCs present in different parts of WJ throughout the length of the UC. For this purpose, using an explant attachment method, WJMSCs were isolated from three different parts of the UC, mainly present towards the placenta (mother part), the center of the whole cord (central part) and the part attached to the fetus (baby part). WJMSCs from all three parts were maintained in normal growth conditions (10% ADMEM) and analyzed for mesenchymal markers, pluripotent genes, proliferation rate and tri-lineage differentiation potential. All WJMSCs were highly proliferative, positively expressed CD90, CD105, CD73 and vimentin, while not expressing CD34, CD45, CD14, CD19 or HLA-DR, differentiated into adipocytes, osteocytes and chondrocytes and expressed pluripotency markers OCT-4, SOX-2 and NANOG at gene and protein levels. Furthermore, MSCs derived from all the parts were shown to have potency towards hepatocyte-like cell differentiation. Human bone marrow-derived MSCs were used as a positive control. Finally, we conclude that WJMSCs derived from all the parts are valuable sources and can be efficiently used in various fields of regenerative medicine.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1007/s00441-017-2699-4) contains supplementary material, which is available to authorized users.</P>

Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

Lee, Won-Jae,Jeon, Ryoung-Hoon,Jang, Si-Jung,Park, Ji-Sung,Lee, Seung-Chan,Baregundi Subbarao, Raghavendra,Lee, Sung-Lim,Park, Bong-Wook,King, William Allan,Rho, Gyu-Jin Hindawi Publishing Corporation 2015 Stem cells international Vol.2015 No.-

<P>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 (NF<SUB>3</SUB>) and optimal number of reference genes (NF<SUB>opt</SUB>). 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 (<I>r</I> > 0.9) between NF<SUB>3</SUB> and NF<SUB>opt</SUB>. Overall, the present study showed that<I> HMBS</I>,<I> YWHAZ</I>,<I> SDHA</I>, and<I> TBP </I>are suitable reference genes for qRT-PCR in porcine MSCs.</P>