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>

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>

Comparison of mesenchymal stem cells isolated from various tissues of isogenic mini-pig

전병균,Dinesh Bharti,이원재,장시정,박지성,정계준,노규진 한국통합생물학회 2015 Animal cells and systems Vol.19 No.6

The present study compared the cellular properties based on cell surface differentiation markers, telomerase activity, stem cell-specific transcripts and differentiation capacity into adipocytes, osteocytes and neurocytes in multipotent mesenchymal stem cells (MSCs) isolated from bone marrow (BMSCs), adipose (ASCs), ovarian (OSCs), muscle (MuSCs) and skin (SSCs) tissues of the same donor mini-pig. Using flow cytometry, all isolated MSCs expressed stem cell-positive surface markers CD29, CD44 and Vimentin, at a high level. Using reverse transcription-polymerase chain reaction, BMSCs, ASCs and SSCs showed higher expression of stem cell-specific transcripts (NANOG, OCT-4 and SOX-2) as compared to OSCs and MuSCs. Telomerase activity was detected by relative-quantitative telomerase repeats amplification protocol at a similar level in all MSCs. Further, analysis of cytochemical staining and lineage-specific transcripts demonstrated that all MSCs get easily differentiated into adipocytes, except for OSCs, whereas, BMSCs and MuSCs easily differentiated into osteocytes, compared to ASCs, OSCs and SSCs. Furthermore, all MSC groups showed the same level of capacity for neurocytes differentiation. Based on these results, the cellular properties were dominantly expressed in BMSCs, ASCs and SSCs, whereas the differentiation capacity was dominantly expressed in BMSCs and MuSCs, compared to others MSCs. Taken together, BMSCs could potentially be used as a good source of MSCs for clinical application and fundamental stem cell research.

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>

Cryopreservation of Human Wharton’s Jelly-derived Mesenchymal Stem Cells Following Controlled Rate Freezing Protocol Using Different Cryoprotectants; A Comparative Study

Shivakumar, Sharath Belame,Bharti, Dinesh,Jang, Si-Jung,Hwang, Sun-Chul,Park, Ji-Kwon,Shin, Jeong-Kyu,Byun, June-Ho,Park, Bong-Wook,Rho, Gyu-Jin Korean Society for Stem Cell Research 2015 International journal of stem cells Vol.8 No.2

<P><B>Objectives</B></P><P>To compare the effect of three different cryoprotectants on basic stem cell characteristics for the possibility of using well defined, dimethyl sulfoxide (DMSO) and serum free freezing solutions to cryopreserve human Wharton’s jelly-derived mesenchymal stem cells (WJMSCs) following controlled rate freezing protocol.</P><P><B>Methods</B></P><P>The mesenchymal stem cells isolated from human Wharton’s jelly were cryopreserved using 10% DMSO, 10% polyvinylpyrrolidone (PVP) and a cocktail solution comprising of 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol following controlled rate freezing protocol. We investigated the post-thaw cell viability, morphology, proliferation capacity, basic stem cell characteristics, in vitro differentiation potential and apoptosis-related gene expression profile before and after cryopreservation.</P><P><B>Results</B></P><P>The cryoprotectant 10% DMSO has shown higher post-thaw cell viability of 81.2±0.58% whereas 10% PVP and cocktail solution have shown 62.87±0.35% and 72.2±0.23%, respectively at 0 h immediately thawing. The cell viability was further reduced in all the cryopreserved groups at 24 h later post-thaw culture. Further, the complete elimination of FBS in cryoprotectants has resulted in drastic reduction in cell viability. Cryopreservation did not alter the basic stem cell characteristics, plasticity and multipotency except proliferation rate. The expression of pro-apoptotic <I>BAX</I> and <I>p53</I> genes were higher whilst <I>p21</I> was lower in all the cryopreserved groups when compare to the control group of WJMSCs.</P><P><B>Conclusion</B></P><P>Although 10% DMSO has shown higher post-thaw cell viability compare to 10% PVP and cocktail solution, the present study indicates the feasibility of developing a well-defined DMSO free cryosolution which can improve storage and future broad range applications of WJMSCs in regenerative medicine without losing their basic stem cell characteristics.</P>

Delay of cell growth and loss of stemness by inhibition of reverse transcription in human mesenchymal stem cells derived from dental tissue

이원철,전병균,김대영,김미정,이현정,Dinesh Bharti,이성호,강영훈,노규진 한국통합생물학회 2019 Animal cells and systems Vol.23 No.5

The present study investigated the cellular properties in the dental tissue-derived mesenchymal stem cells (DSCs) exposed to nevirapine (NVP), an inhibitor of reverse transcriptase (RTase). After a prolonged exposure of DSCs for 2 weeks, the population doubling time (PDT) was significantly (P < .05) increased by delayed cell growth in the DSCs treated with 250 and 500 μM NVP, compared with untreated DSCs. Furthermore, the G1 phase of cell cycle with high activity of senescence-associated β-galactosidase was also significantly (P < .05) increased in the 250 μM NVP-treated DSCs, compared with untreated DSCs. The level of telomerase activity was unchanged between control and treatment. However, following the treatment of NVP, negative surface markers for mesenchymal stem cells (MSCs), such as CD34 and CD45, were significantly (P < .05) increased, while positive surface markers for MSCs, such as CD90 and CD105, were significantly (P < .05) decreased in the NVP-treated DSCs than those of untreated DSCs. Furthermore, the differentiation capacity into mesodermal lineage was gradually decreased, and a significant (P < .05) decrease of expression level of NANOG, OCT-4 and SOX-2 transcripts was observed in the DSCs treated with NVP, compared with untreated control DSCs. Taken together, the present results have revealed that inhibition of RTase by NVP induces delayed cell growth and loss of stemness.

Comparative analysis of three different protocols for cholinergic neuron differentiation in vitro using mesenchymal stem cells from human dental pulp

강영훈,Sharath Belame Shivakumar,손영범,Dinesh Bharti,장시정,허강선,박원욱,변준호,박봉욱,노규진 한국통합생물학회 2019 Animal cells and systems Vol.23 No.4

A decrease in the activity of choline acetyltransferase, the enzyme responsible for acetylcholine synthesis in the cholinergic neurons cause neurological disorders involving a decline in cognitive abilities, such as Alzheimer’s disease. Mesenchymal stem cells (MSCs) can be used as an efficient therapeutic agents due to their neuronal differentiation potential. Different source derived MSCs may have different differentiation potential under different inductions. Various in vitro protocols have been developed to differentiate MSCs into specific neurons but the comparative effect of different protocols utilizing same source derived MSCs, is not known. To address this issue, dental pulp derived MSCs (DPSCs) were differentiated into cholinergic neurons using three different protocols. In protocol I, DPSCs were pre-induced with serum-free ADMEM containing 1 mM of β-mercaptoethanol for 24 h and then incubated with 100 ng/ml nerve growth factor (NGF) for 6 days. Under protocol II, DPSCs were cultured in serum-free ADMEM containing 15 μg/ ml of D609 (tricyclodecan-9-yl-xanthogenate) for 4 days. Under protocol III, the DPSCs were cultured in serum-free ADMEM containing 10 ng/ml of basic fibroblast growth factor (bFGF), 50 μM of forskolin, 250 ng/ml of sonic hedgehog (SHH), and 0.5 μM of retinoic acid (RA) for 7 days. The DPSCs were successfully trans-differentiated under all the protocols, exhibited neuronlike morphologies with upregulated cholinergic neuron-specific markers such as ChAT, HB9, ISL1, BETA-3, and MAP2 both at mRNA and protein levels in comparison to untreated cells. However, protocol III-induced cells showed the highest expression of the cholinergic markers and secreted the highest level of acetylcholine.

Terminal differentiation into adipocyte and growth inhibition by PPARγ activation in human A549 lung adenocarcinoma cells

김대영,Sun-Ha Moon,한장호,김미정,오성주,BHARTI DINESH,이성호,박종근,노규진,전병균 한국통합생물학회 2020 Animal cells and systems Vol.24 No.6

The present study investigated the terminal differentiation capacity into adipocytes and subsequent growth inhibition in A549 cancer cells treated with pioglitazone (PGZ), a PPARγ activator. The rate of cell growth in A549 cells was significantly (P < .05) inhibited in concentrations above 10 μM PGZ while maintaining less cytotoxic effects in MRC-5 fibroblasts. Following 50 μM PGZ treatment, population doubling time (PDT) was significantly (P < .05) increased by inhibition of cell growth, as per increasing PGZ exposure time by up to 4 weeks. The adiposome-like vesicles were commonly observed in the PGZ-treated A549 cells, and the vesicles were highly stained with Oil-Red O solution. In addition, the cell size and expression of GLUT4 and PPARγ were significantly (P < .05) increased, as per increasing PGZ exposure time by up to 4 weeks. The significant (P < .05) down-regulation of telomerase activity and up-regulation of senescence-associated β-galactosidase (SA β-GAL) activity was displayed in the PGZ-treated A549 cells, as per increasing PGZ exposure time by up to 4 weeks. The G1 phase of the cell cycle was also significantly (P < .05) increased in the PGZ-treated A549 cells compared with untreated A549 cells. The present results have demonstrated that activation of PPARγ using PGZ induces cellular differentiation into adipocytes and inhibits cell growth in the A549 cancer cells. The terminal differentiation into adipocytes could offer potent chemotherapy in the cancer cells showing high glucose metabolism.