Micromanipulation of culture niche permits long-term expansion of dental pulp stem cells--an economic and commercial angle.

Stem cells isolated from dental pulp possess the capacity for self-renewal and the potential for multi-lineage differentiation. However, dental pulp stem cells have different characteristics in terms of their culture conditions. The success of stem cells culture is governed by its micro-environmental niche. Therefore, we studied the effects of culture niche on long-term expansion of dental pulp stem cells in terms of cell morphology, growth kinetics, senescence pattern, cell surface marker expression differentiation capacity, and seeding plating density of dental pulp stem cells in four different, widely used media composition Among the various basal media tested, α-minimum essential media and knock out-minimum essential media supplemented with 10% fetal bovine serum were found to be the most optimal media composition in preserving the phenotypic characteristics and differentiation potential for prolonged periods as compared with DMEM-F12 and DMEM-LG. Plating density has been shown to affect overall yield. As a conclusion, the adoption of an appropriate culture system significantly improved cell yield, thus enabling the attainment of sufficient yields for therapeutic applications economizing in terms of cost of production and minimizing seeding cell density for maximum yield.

Establishment of a brain tumor tissue repository in India: maintaining quality standards.

Tumor tissue repositories (TTRs) play a pivotal role in both basic and translational research by acting as a conduit to facilitate innovative research, thereby providing solutions to treat the incurable disease--'Cancer'. One of the fundamental requirements to achieve this goal would be the acquisition of high quality tumor tissue specimens that are stored in such a manner that its integrity is preserved. Further, a quality system should be in place that assures the compliance of procedures that are the key to a smooth functioning of all the inter-related departments that play a key role in the entire operations. To address this, we have initiated an effort to build a tumor tissue repository of brain tumor tissues in the Southern part of the Indian sub-continent. One of the cardinal features of brain tumors is the heterogeneity, both phenotypically and genotypically. Moreover, significant gaps exist in current understanding of the molecular pathways involved in the genesis, progression, and biological and clinical behavior of brain tumors. We hope that our initiative will provide researchers accessibility to a reserve of high quality tissues in this part of the globe. We have created and validated a complete histology service including tissue processing, embedding, sectioning and H&E staining for fixed tissues, in addition to creating and staining frozen sections. To our knowledge, such a structured initiative to store brain tumor samples is the first of its kind in the India.

Rho kinase inhibitor y27632 alters the balance between pluripotency and early differentiation events in human embryonic stem cells.

Human embryonic stem cells (hESC) differentiate spontaneously in culture and develop a complex microenvironment comprising of autologously derived niche that in turn supports their pluripotency. The basic hypothesis that we deal with is that hESCs undergoing differentiation, sequentially generate trophectoderm and endoderm lineages and thereafter influence further events through the production of growth factors. These factors control the fate of hESCs either by promoting or retarding the recruitment of new cells in the differentiation program. This scenario therefore represents an analog of the in vivo situation in which extra-embryonic tissues influence the behavior of the inner cell mass (ICM). The premise of the paper is the Rho kinase inhibitor Y27632 that can spatiotemporally alter this balance between pluripotency and differentiation. To evaluate the composition and inclination of lineage specification during spontaneous differentiation, we have studied the hESC colonies and their surrounding niche as interdependent entities. We show that the population of fibroblastic niche that surrounds hESC colonies co-expresses trophectoderm and niche cell markers including SSEA1, hCG, progesterone, HAND1, pSmad1 and FGFR1 as early as day 4. A sudden increase in the expression of GATA4 and AFP secretion indicated putative endoderm formation on day 6 in both control and Y27632 treated cultures. On day 6, 20 microM of Y27632 supplementation significantly reduced the trophectoderm-like niche population without affecting endoderm formation, enhanced the average size and number of hESC colonies, decreased IGF1 secretion thereby improving the pluripotency. Overall our findings support the afore mentioned hypothesis and demonstrate that closely packed epithelial trophectoderm-like cells bordering the hESC colonies present an initial and imminent localized niche which is spatiotemporally regulated. Such advances in understanding the behavior and modulation of hESC and its surrounding niche would facilitate better differentiation protocols for applications in regenerative medicine and drug screening.

Y-27632 enhances differentiation of blastocyst like cystic human embryoid bodies to endocrinologically active trophoblast cells on a biomimetic platform.

Trophoblast differentiation and formation of the placenta are important events linked to post-implantation embryonic development. Models mimicking the biology of trophoblast differentiation in a post-implantation maternal microenvironment are needed for understanding disorders like placental-ischemia or for applications in drug-screening, and would help in overcoming the ethical impasse on using human embryos for such research. Here we attempt to create such a model by using embryoid bodies (EBs) and a biomimetic platform composed of a bilayer of fibronectin and gelatin on top of low-melting agarose. Using this model we test the hypothesis that cystic-EBs (day 30) that resemble blastocysts morphologically, are better sources as compared to noncytic EBs (day 10), for functional trophoblast differentiation; and that the Rho kinases inhibitor Y27632 can enhance this differentiation. Non/cytic EBs with/out Y27632 were grown on this platform for 28 days, and screened from secretion and expression of trophoblast and other lineage markers using ECLIA, RT-PCR, and Immunofluorescence. All EBs attached on this surface and rapidly proliferated into hCG and progesterone (P2) secreting functional trophoblast cells. However, the cells derived from cytic-EBs and cytic-EBs+ Y27632 showed the maximum secretion of these hormones and expressed IGF2, supporting our hypothesis. Also Y27632 reduced extraembryonic endoderm and trophoblast lineage differentiation from early noncystic-EBs, whereas, it specifically enhanced the induction of trophoblast and multinucleated syncitiotrophoblast differentiation from late cystic-EBs. In vivo trophoblast differentiation can be replicated in fibronectin based biomaterials, using cytic-EBs and by maneuvering the Rho-ROCK pathways. Response of EBs to a compound may vary temporally, and determination of their right stage is crucial for applications in directed-differentiation or drug-screening.

Propensity of human embryonic stem cell lines during early stage of lineage specification controls their terminal differentiation into mature cell types.

Human embryonic stem cells (hESCs) are able to stably maintain their characteristics for an unlimited period; nevertheless, substantial differences among cell lines in gene and protein expression not manifested during the undifferentiated state may appear when cells differentiate. It is widely accepted that developing an efficient protocol to control the differentiation of hESCs will enable us to produce adequate numbers of desired cell types with relative ease for diverse applications ranging from basic research to cell therapy and drug screening. Hence of late, there has been considerable interest in understanding whether and how hESC lines are equivalent or different to each other in their in vitro developmental tendencies. In this study, we compared the developmental competences of two hESC lines (HUES-9 and HUES-7) at molecular, cellular and functional levels, upon spontaneous differentiation without any added inducing agents. Both cell lines generated the three embryonic germ layers, extra-embryonic tissues and primordial germ cells during embryoid body (EB) formation. However HUES-9 showed a stronger propensity towards formation of neuroectodermal lineages, whereas HUES-7 differentiated preferentially into mesoderm and endoderm. Upon further differentiation, HUES-9 generated largely neural cells (neurons, oligodendrocytes, astrocytes and gangliosides) whereas HUES-7 formed mesendodermal derivatives, including cardiomyocytes, skeletal myocytes, endothelial cells, hepatocytes and pancreatic cells. Overall, our findings endorse the hypothesis that independently-derived hESCs biologically differ among themselves, thereby displaying varying differentiation propensity. These subtle differences not only highlight the importance of screening and deriving lines for lineage-specific differentiation but also indicate that individual lines may possess a repertoire of capabilities that is unique.

Adult stem cells: a clinical update.

There has been unprecedented interest in stem cell research mainly because of their true potential and hope that they offer to the patients as a cell therapy with the prospect to treat hitherto incurable diseases. Despite the worldwide interest and efforts that have been put in this research, major fundamental issues are still unresolved. Adult stem cells such as hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) are already under clinical applications and there are several examples of plasticity and self-renewal where adult stem cells or their precursor cells can be re-programmed by extra cellular cues or internal cues to alter their character in a way that could have important application for cell therapy and regenerative medicine. From a clinical perspective, no other area of stem cell biology has been applied as successfully as has transplantation of bone marrow stem cells and cord blood stem cells for the treatment of hematological diseases. In the last few years, research in stem cell biology has expanded staggeringly, engendering new perspectives concerning the identity, origin, and full therapeutic potential of tissue-specific stem cells. This review will focus on the use of adult stem cells, its biology in the context of cell plasticity and their therapeutic potential for repair of different tissues and organs.