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ES Cell Differentiation (e + cell_differentiation)
Selected AbstractsChanges in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin-producing cells in vitro and in vivoDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2009Ortwin Naujok Abstract Background Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type 1 diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation. Methods ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis. Results In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the co-stored IAPP as well as the glucose recognition structures. In contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol. Conclusions A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation. Copyright © 2009 John Wiley & Sons, Ltd. [source] Generation of hepatocytes from cultured mouse embryonic stem cellsLIVER TRANSPLANTATION, Issue 10 2003Xiao Ling Kuai Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of fertilized blastocysts in vitro. ES cells can be induced to undergo differentiation into potentially all cell types. The aim of this study is to examine the differentiating potential of mouse ES cells into hepatocytes in the presence of retinoic acid (RA), hepatocyte growth factor (HGF), and ,-nerve growth factor (,-NGF). RA, HGF, and ,-NGF were added to the cell culture. Hepatocyte induction was confirmed morphologically, as well as biochemically, through immunohistochemical assays of ,1 -antitrypsin (,1 -AT) and alfafetaprotein (AFP) expression and reverse-transcriptase polymerase chain reaction tests for the presence of albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SAPK/ERK kinase-1 (SEK1) messenger RNA, produced only by functioning hepatocytes. Fifteen days after the addition of HGF and ,-NGF to the cell culture, many epithelioid cells were noticed. ,1 -AT, AFP, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SEK1 messenger RNA expression also was detected, indicating successful ES cell differentiation into functioning hepatocytes. However, in the presence of RA alone, only transthyretin messenger RNA was positive, whereas no other expression pertaining to functioning hepatocytes could be detected. In the presence of HGF and ,-NGF, mouse ES cells can differentiate into functioning hepatocytes, whereas RA function is limited. [source] Enhanced differentiation of embryonic stem cells using co-cultivation with hepatocytesBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2008Rebecca N. Moore Abstract We examined the effects of co-cultivated hepatocytes on the hepatospecific differentiation of murine embryonic stem (ES) cells. Utilizing an established mouse ES cell line expressing high or low levels of E-cadherin, that we have previously shown to be responsive to hepatotrophic growth factor stimulation (Dasgupta et al., 2005. Biotechnol Bioeng 92(3):257,266), we compared co-cultures of cadherin-expressing ES (CE-ES) cells with cultured rat hepatocytes, allowing for either paracrine interactions (indirect co-cultures) or both juxtacrine and paracrine interactions (direct co-cultures, random and patterned). Hepatospecific differentiation of ES cells was evaluated in terms of hepatic-like cuboidal morphology, heightened gene expression of late maturation marker, glucose-6-phosphatase in relation to early marker, alpha-fetoprotein (AFP), and the intracellular localization of albumin. Hepatocytes co-cultured with growth factor primed CE-ES cells markedly enhanced ES cell differentiation toward the hepatic lineage, an effect that was reversed through E-cadherin blockage and inhibited in control ES cells with reduced cadherin expression. Comparison of single ES cell cultures versus co-cultures show that direct contact co-cultures of hepatocytes and CE-ES cells maximally promoted ES cell commitment towards hepatodifferentiation, suggesting cooperative effects of cadherin-based juxtacrine and paracrine interactions. In contrast, E-cadherin deficient mouse ES (CD-ES) cells co-cultured with hepatocytes failed to show increased G6P expression, confirming the role of E-cadherin expression. To establish whether albumin expression in CE-ES cells was spatially regulated by co-cultured hepatocytes, we co-cultivated CE-ES cells around micropatterned, pre-differentiated rat hepatocytes. Albumin localization was enhanced "globally" within CE-ES cell colonies and was inhibited through E-cadherin antibody blockage in all but an interfacial band of ES cells. Thus, stem cell based cadherin presentation may be an effective tool to induce hepatotrophic differentiation by leveraging both distal/paracrine and contact/juxtacrine interactions with primary cells of the liver. Biotechnol. Bioeng. © 2008 Wiley Periodicals, Inc. [source] Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process controlBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2005Magnus Schroeder Abstract It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5,×,106 cells/mL after 9 days of differentiation. Approximately 30%,40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (,MHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28,×,109 cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications. © 2005 Wiley Periodicals, Inc. [source] Supplementation-dependent differences in the rates of embryonic stem cell self-renewal, differentiation, and apoptosisBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2003Sowmya Viswanathan Abstract Although it is known that leukemia inhibitory factor (LIF) supports the derivation and expansion of murine embryonic stem (ES) cells, it is unclear whether this is due to inhibitory effects of LIF on ES cell differentiation or stimulatory effects on ES cell survival and proliferation. Using an ES cell line transgenic for green fluorescent protein (GFP) expression under control of the Oct4 promoter, we were able to simultaneously track the responses of live Oct4-GFP-positive (ES) and -negative (differentiated) fractions to LIF, serum, and other growth factors. Our findings show that, in addition to inhibiting differentiation of undifferentiated cells, the administration of LIF resulted in a distinct dose-dependent survival and proliferation advantage, thus enabling the long-term propagation of undifferentiated cells. Competitive responses from the differentiated cell fraction could only be elicited upon addition of serum, fibroblast growth factor-4 (FGF-4), or insulin-like growth factor-1 (IGF-1). The growth factors did not induce additional differentiation of ES cells, but rather they significantly improved the proliferation of already differentiated cells. Our analyses show that, by adjusting culture conditions, including the type and amount of growth factors or cytokines present, the frequency of media exchange, and the presence or absence of serum, we could selectively and specifically alter the survival, proliferation, and differentiation dynamics of the two subpopulations, and thus effectively control population outputs. Our findings therefore have important applications in engineering stem cell culture systems to predictably generate desired stem cells or their derivatives for various regenerative therapies. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng84: 505,517, 2003. [source] | ||||||||||