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Klattenhoff C.A.,Massachusetts Institute of Technology | Scheuermann J.C.,Massachusetts Institute of Technology | Surface L.E.,Massachusetts Institute of Technology | Bradley R.K.,Massachusetts Institute of Technology | And 13 more authors.
Cell | Year: 2013

Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development. © 2013 Elsevier Inc.


Baharvand H.,Royan Institute for Stem Cell Biology and Technology | Baharvand H.,University of Tehran | Salekdeh G.H.,Royan Institute for Stem Cell Biology and Technology | Salekdeh G.H.,Agricultural Biotechnology Research Institute of Iran | And 2 more authors.
Nature Protocols | Year: 2010

Here we describe a simple and efficient human embryonic stem (ES) and induced pluripotent stem (iPS) cells cryopreservation protocol. This protocol involves the use of Rho-associated kinase (ROCK) inhibitor, Y-27632, for the feeder-free dissociated cells. The addition of ROCK inhibitor to both pre- and post-thaw culture media enhanced the cloning efficiency. The presence of Y-27632 in Matrigel further increased the cloning efficiency. As compared with other available protocols for human ES and iPS cells cryopreservation, our protocol differs in the technical simplicity, high cloning efficiency and post-thawing passaging. We believe that this protocol could be a generally applicable and robust platform for feeder-free cryopreservation and the expansion of present and future applications of human ES and iPS cells. The treatment with ROCK inhibitor, cell harvesting and the freezing-thawing process usually takes about 2 h excluding overnight incubation at -80 °C. © 2010 Nature Publishing Group.


Moradi S.,Royan Institute for Stem Cell Biology and Technology | Moradi S.,University of Tehran | Asgari S.,University of Queensland | Baharvand H.,Royan Institute for Stem Cell Biology and Technology | Baharvand H.,University of Tehran
Stem Cells | Year: 2014

It is now well-established that somatic cells can be reprogrammed to alternative cell fates by ectopic coexpression of defined factors. Reprogramming technology has uncovered a huge plasticity besides gene regulatory networks (GRNs) of differentiated cell states. MicroRNAs (miRNAs), which are an integral part of GRNs, have recently emerged as a powerful reprogramming toolbox. They regulate numerous genes, thereby modulating virtually all cellular processes, including somatic cell reprogramming. Not only can miRNAs provide novel opportunities for interrogating mechanisms of induced pluripotency and direct lineage reprogramming but they also offer hope for the efficient creation of safe cell sources for regenerative medicine. In reviewing landmark roles of miRNAs in cell reprogramming, we offer suggestions for evolution of the reprogramming field.© AlphaMed Press 2013.


Zangeneh M.,Royan Institute for Stem Cell Biology and Technology
Obstetrics and Gynecology | Year: 2010

Background: Pyomyoma (suppurative leiomyoma) is a rare disease that is a serious complication. Most cases have occurred in pregnant or postmenopausal women. Case: A perimenopausal woman presented with fever and shoulder pain. She had no predisposing factors or history of leiomyoma. Ultrasonographic as well as abdominal and pelvic computed tomography scans showed an enlarged uterus with two large masses. Internal heterogeneous echogenicity was noted in the lower segment and body of the uterus. The elevated temperature continued despite a 3-day antibiotic course of clindamycin, ceftriaxone, and gentamicin. With a clinical impression of infected leiomyoma, she underwent a total abdominal hysterectomy and bilateral salpingo-oophorectomy. Pathological findings showed a leiomyoma with abscess formation. The patient responded well to surgery. Conclusion: Pyomyoma may be difficult to diagnose, especially in women with a nonspecific clinical presentation. Delayed diagnosis may result in serious complications, and surgery and broad spectrum antibiotics are indicated. © 2010 by The American College of Obstetricians and Gynecologists. Published by Lippincott Williams & Wilkins.


Abbasalizadeh S.,Royan Institute for Stem Cell Biology and Technology | Baharvand H.,Royan Institute for Stem Cell Biology and Technology | Baharvand H.,University of Tehran
Biotechnology Advances | Year: 2013

Recent technological advances in the generation, characterization, and bioprocessing of human pluripotent stem cells (hPSCs) have created new hope for their use as a source for production of cell-based therapeutic products. To date, a few clinical trials that have used therapeutic cells derived from hESCs have been approved by the Food and Drug Administration (FDA), but numerous new hPSC-based cell therapy products are under various stages of development in cell therapy-specialized companies and their future market is estimated to be very promising. However, the multitude of critical challenges regarding different aspects of hPSC-based therapeutic product manufacturing and their therapies have made progress for the introduction of new products and clinical applications very slow. These challenges include scientific, technological, clinical, policy, and financial aspects. The technological aspects of manufacturing hPSC-based therapeutic products for allogeneic and autologous cell therapies according to good manufacturing practice (cGMP) quality requirements is one of the most important challenging and emerging topics in the development of new hPSCs for clinical use. In this review, we describe main critical challenges and highlight a series of technological advances in all aspects of hPSC-based therapeutic product manufacturing including clinical grade cell line development, large-scale banking, upstream processing, downstream processing, and quality assessment of final cell therapeutic products that have brought hPSCs closer to clinical application and commercial cGMP manufacturing. © 2013 Elsevier Inc.


Eslaminejad M.B.,Royan Institute for Stem Cell Biology and Technology | Fallah N.,University of Tehran
Iranian Journal of Medical Sciences | Year: 2014

Background: Hyaline cartilage defects exhibit a major challenge in the field of orthopedic surgery owing to its limited repair capacity. On the other hand, mesenchymal stem cells (MSCs) are regarded as potent cells with a property of cartilage regeneration. We aimed to optimize marrow-derived MSC chondrogenic culture using a small bioactive molecule referred to as BIO. Methods: MSCs from the marrow of NMRI mice were extracted, culture-expanded, and characterized. Micro-mass culture was then established for chondrogenic differentiation (control group). The cultures of MSC in chondrogenic medium supplemented with 0.01, 0.05, 0.1, and 1 μM BIO were taken as the experimental groups. Cartilage differentiation was examined by both histological sections and real-time PCR for Sox9, aggrecan, and collagen II at different time points. Moreover, the involvement of the Wnt pathway was investigated. Results: Based on histological sections, there was seemingly more intense metachromatic matrix produced in the cultures with 0.01 μM BIO. In this experimental group, cartilage-specific genes tended to be upregulated at day 14 compared to day 21 of the control group, indicating the accelerating effect of BIO on cartilage differentiation. Overall, there was statistically a significant increase (P=0.01) in the expression level of cartilagespecific genes in cultures with 0.01 μM BIO (enhancing effects). These upregulations appeared to be mediated through the Wnt pathway evident from the significant upregulation of T-cell factor and beta-catenin molecules (P=0.01). Conclusion: Taken together, BIO at 0.01 μM could accelerate and enhance in vitro chondrogenesis of mouse marrow-derived MSCs.


Zomorodian E.,Royan Institute for Stem Cell Biology and Technology | Baghaban Eslaminejad M.,Royan Institute for Stem Cell Biology and Technology
Stem Cells International | Year: 2012

While small bone defects heal spontaneously, large bone defects need surgical intervention for bone transplantation. Autologous bone grafts are the best and safest strategy for bone repair. An alternative method is to use allogenic bone graft. Both methods have limitations, particularly when bone defects are of a critical size. In these cases, bone constructs created by tissue engineering technologies are of utmost importance. Cells are one main component in the manufacture of bone construct. A few cell types, including embryonic stem cells (ESCs), adult osteoblast, and adult stem cells, can be used for this purpose. Mesenchymal stem cells (MSCs), as adult stem cells, possess characteristics that make them good candidate for bone repair. This paper discusses different aspects of MSCs that render them an appropriate cell type for clinical use to promote bone regeneration. © 2012 Elham Zomorodian and Mohamadreza Baghaban Eslaminejad.


Farrokhi A.,Royan Institute for Stem Cell Biology and Technology
Cellular and molecular biology (Noisy-le-Grand, France) | Year: 2012

Reverse transcription quantitative PCR (RT—qPCR) is one of the best methods for the study of mesenchymal stem cell (MSC) differentiation by gene expression analysis. This technique needs appropriate reference or housekeeping genes (HKGs) to normalize the expression of the genes of interest. In the present study the expression stability of six widely used HKGs including Actb, Btub, Hprt, B2m, Gusb and Tfrc was investigated during rat MSC differentiation into osteocytes, adipocytes and chondrocytes lineages using geNorm and NormFinder software. RT—qPCR data analyzed by geNorm revealed the different sets of suitable reference genes for each cell type. NormFinder also showed similar results. Analysis of the combined data of MSCs with each differentiated cell type revealed the considerable shift in expression of some reference genes during differentiation; for example Gusb and B2m were among the least stable genes in MSCs but the most stable in chondrocytes. Normalization of specific genes for each lineage by different reference genes showed considerable difference in their expression fold change. In conclusion, for the appropriate analysis of gene expression during rat MSC differentiation and also for monitoring differentiation procedures, it is better to consider precisely the reference gene stability and select suitable reference genes for each purpose.


Larijani M.R.,Royan Institute for Stem Cell Biology and Technology
Stem cells and development | Year: 2011

Traditionally, undifferentiated pluripotent human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) have been expanded as monolayer colonies in adhesion culture, both in the presence or absence of feeder cells. However, the use of pluripotent stem cells poses the need to scale-up current culture methods. Herein, we present the cultivation of 2 hESC lines (Royan H5 and Royan H6) and 2 hiPSC lines (hiPSC1 and hiPSC4) as carrier-free suspension aggregates for an extended period of time. The cells proliferated over multiple passages kept a stable karyotype, which successfully maintained an undifferentiated state and pluripotency, as determined by marker expressions in addition to in vitro spontaneous and directed differentiation. Additionally, these cells can be easily frozen and thawed without losing their proliferation, karyotype stability, and developmental potential. Transcriptome analysis of the 3 lines revealed that the adherent culture condition was nearly identical to the suspension culture in Royan H5 and hiPSC1, but not in Royan H6. It remains unclear whether this observation at the transcript level is biologically significant. In comparison with recent reports, our study presents a low-cost procedure for long-term suspension expansion of hESCs and hiPSCs with the capability of freeze/thawing, karyotype stability, and pluripotency. Our results will pave the way for scaled up expansion and controlled differentiation of hESCs and hiPSCs needed for cell therapy, research, and industrial applications in a bioreactor culture system.


Patent
Royan Institute for Stem Cell Biology and Technology | Date: 2014-08-19

The various embodiments herein provide a method for derivation and long term establishment of ground state pluripotent embryonic stem cells. Further the embodiments herein provides a method to inhibit the ERK and TGF signalling pathways for long term maintenance of the embryonic stem cells. The R2i mouse embryonic stem (ES) cells are derived from 3.5 day blastocysts. The mouse ES cells are cultured in media containing R2i and 2i inhibitors of ERK and TGF pathways. The ES cells are subjected to in vitro and in vivo differentiation. The ES cells are subjected to RT-PCR and qRT-PCR, flow cytometry and karyotyping. The result reveals that the R2i maintains the ground state of ES cells and self renewal. Also R2i increases embryonic cleavage and clonal propagation of ES. Further R2i asserts genomic integrity and pluripotency of ES.

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