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Research Containing: Myocytes

Efficient Differentiation of Cardiomyocytes from Human Pluripotent Stem Cells with Growth Factors

by on 22 August 2016in Biology & Biotechnology No comment

Human pluripotent stem cells have tremendous replicative capacity and demonstrated potential to generate functional cardiomyocytes. These cardiomyocytes represent a promising source for cell replacement therapy to treat heart disease and may serve as a useful tool for drug discovery and disease modeling. Efficient cardiomyocyte differentiation, a prerequisite for the application of stem cell-derived cardiomyocytes, can be achieved with a growth factor-guided method. Undifferentiated cells are sequentially treated with activin A and BMP4 in a serum-free and insulin-free medium and then maintained in a serum-free medium with insulin. This method yields as much as >75 % cardiomyocytes in the differentiation culture within 2 weeks, and the beating cardiomyocytes have expected molecular, cellular, and electrophysiological characteristics. In this chapter, we describe in detail the differentiation protocol and follow-up characterization focusing on immunocytochemistry, quantitative RT-PCR, and flow cytometry analysis.

Related URLs:
http://www.ncbi.nlm.nih.gov/pubmed/25836579

Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning, lateral vessel (STLV) bioreactor

by on 9 June 2015in Biology & Biotechnology No comment

Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated. Using 3 x 10(5) cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.

Related URLs:
<Go to ISI>://WOS:000293752000009

Pretreatment of Rat Bone Marrow Mesenchymal Stem Cells with a Combination of Hypergravity and 5-Azacytidine Enhances Therapeutic Efficacy for Myocardial Infarction

by on 9 June 2015in Biology & Biotechnology No comment

Background and Purpose: The in vivo cardiac differentiation and functional effects of unmodified adult bone marrow mesenchymal stem cells (BMSCs) after myocardial infarction (MI) is controversial. Our previous results suggested that hypergravity promoted the cardiomyogenic differentiation of BMSCs, and thus we postulated that ex vivo pretreatment of BMSCs using hypergravity and 5-azacytidine (5-Aza) would lead to cardiomyogenic differentiation and result in superior biological and functional effects on cardiac regeneration of infarcted myocardium. Methods: We used a rat MI model generated by ligation of the coronary artery. Homogeneous rat BMSCs were isolated, culture expanded, and differentiated into a cardiac lineage by adding hypergravity (2G) for 3 days and 5-Aza (50 lmol/L, 24 h). Rats underwent BMSCs (labeled with DAPI) injection after the infarction and were randomized into five groups. Group A rats received the control medium, Group B rats received unmodified BMSCs, Group C rats received BMSCs treated with hypergravity, Group D rats received BMSCs treated with 5-Aza, and Group E rats received BMSCs treated with 5-Aza and hypergravity (n = 6). Results: After hypergravity and 5-Aza treatment, BMSCs showed positive for the early muscle and cardiac markers GATA-4, MEF-2, and Nkx2-5 with RTPCR. We also found that hypergravity could enhance the activities of MEF-2 via promoting the nuclear export of HDAC5. The frozen section showed that the implanted BMSCs labeled with DAPI survived and angiogenesis was identified at the implantation site. In Groups B, C, D, and E rats, pre-treated BMSCs colocalized with alpha-actinin, and Group E rats showed a significantly larger increase in left ventricular function. Conclusions: The biological ex vivo cardiomyogenic differentiation of adult BMSCs with hypergravity and 5-Aza prior to their transplantation is feasible and appears to improve their in vivo cardiac differentiation as well as the functional recovery in a rat model of the infarcted myocardium. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 473-482, 2011

Related URLs:
<Go to ISI>://WOS:000289431600022

In vitro osteogenic differentiation of rat mesenchymal stem cells in a microgravity bioreactor

by on 9 June 2015in Biology & Biotechnology No comment

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to differentiate into osteoblasts, chondroblasts, myocytes, and adipocytes. They have potential for bone tissue engineering by the utilization of in vitro expanded cells with osteogenic capacity and their delivery to the appropriate sites via biomaterial scaffolds. The objective was to evaluate the potential of rat bone marrow MSCs to form 3D bone-like tissue by the use of mineralized poly(DL-lactic-co-glycolic acid) (PLGA) foam and osteoinductive medium under rotating culture conditions. PLGA foams were prepared by solvent casting and particulate leaching, then mineralized by incubating in simulated body fluid. MSCs isolated from the bone marrow of young Wistar rats were expanded and seeded on the mineralized scaffolds. The cell-polymer constructs were then cultured in a slow turning lateral vessel-type rotating bioreactor for 4 weeks under the effect of osteogenic inducers, b-glycerophosphate, ascorbic acid and dexamethasone. Mineralization was evaluated using FT-IR and increases in dry mass; morphology changes of the mineralized foams and cell adhesion was characterized by SEM; cell viability was monitored by MTT (3-(4,5-dimethylthia-zol-2-yl-2,5-diphenyl tetrazolium bromide). Osteogenic differentiation was determined by using immunohistochemistry (anti-Osteopontin). Results indicate the feasibility of bone tissue engineering with MSCs and mineralized PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures under appropriate bioreactor conditions.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed8&AN=2008202967
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1177%2F0883911508091828&issn=0883-9115&isbn=&volume=23&issue=3&spage=244&pages=244-261&date=2008&title=Journal+of+Bioactive+and+Compatible+Polymers&atitle=In+vitro+osteogenic+differentiation+of+rat+mesenchymal+stem+cells+in+a+microgravity+bioreactor&aulast=Koc&pid=%3Cauthor%3EKoc+A.%3C%2Fauthor%3E&%3CAN%3E2008202967%3C%2FAN%3E

Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells

by on 9 June 2015in Biology & Biotechnology No comment

BACKGROUND: Stem cell therapy has emerged as a potential therapeutic option for tissue engineering and regenerative medicine, but many issues remain to be resolved, such as the amount of seed cells, committed differentiation and the efficiency. Several previous studies have focused on the study of chemical inducement microenvironments. In the present study, we investigated the effects of gravity on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into force-sensitive or force-insensitive cells. METHODS AND RESULTS: Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and beta-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes). CONCLUSION: Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed9&AN=19772591
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:19772591&id=doi:&issn=1423-0127&isbn=&volume=16&issue=&spage=87&pages=87&date=2009&title=Journal+of+biomedical+science&atitle=Gravity%2C+a+regulation+factor+in+the+differentiation+of+rat+bone+marrow+mesenchymal+stem+cells&aulast=Huang&pid=%3Cauthor%3EHuang+Y.%3C%2Fauthor%3E&%3CAN%3E19772591%3C%2FAN%3E

Computational modeling for the optimization of a cardiogenic 3D bioprocess of encapsulated embryonic stem cells

by on 9 June 2015in Biology & Biotechnology No comment

We present a computational fluid dynamics (CFD)-based model aimed at the identification of optimized culture conditions promoting efficient cardiogenesis of hydrogel-bead-encapsulated embryonic stem cells (ESCs) within a rotating bioreactor. The numerical approach, integrating diffusion, convection, and multiphase fluid dynamics calculations, allowed to evaluate (i) the microgravity motion of the floating beads, (ii) the O-2 delivery to the cells, also (iii) taking into account the cellularO(2) consumption, as a function of different rotation speeds of the breeding chamber. According to our results, a 25rpm rotation (i) enhances an adequate mixing of the cell carriers, avoiding sedimentation and excessive packing, also maintaining a quite homogeneous distribution of the suspended beads and (ii) imparts a proper cellular O-2 supply, providing cells close to a normoxia condition. The bioreactor working conditions derived from the numerical analysis allowed the attainment of in vitro long-term cell viability maintenance, supporting efficient large-scale generation of ESC-derived cardiomyocytes (ESC-DCs) through a chemical-based conditioning bioprocess. In conclusion, we demonstrated the feasibility of using CFD-based tools, as a reliable and cost-effective strategy to assist the design of a 3D cardiogenic bioprocess.

Related URLs:
<Go to ISI>://WOS:000300230600020