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Murine Embryonic Stem (murine + embryonic_stem)

Distribution by Scientific Domains

Life Sciences	75%

Selected Abstracts

Characterisation of a P140K mutant O6 -methylguanine-DNA-methyltransferase (MGMT)-expressing transgenic mouse line with drug-selectable bone marrow

THE JOURNAL OF GENE MEDICINE, Issue 9 2006
Belinda A. Kramer
Abstract Background Gene transfer of the P140K mutant of O6 -methylguanine-DNA-methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) provides a mechanism for drug resistance and the selective expansion of gene-modified cells in vivo. Possible clinical applications for this strategy include chemoprotection to allow dose escalation of alkylating chemotherapy, or combining MGMT(P140K) expression with a therapeutic gene in the treatment of genetic diseases. Our aim is to use MGMT(P140K)-driven in vivo selection to develop allogeneic micro-transplantation protocols that rely on post-engraftment selection to overcome the requirement for highly toxic pre-transplant conditioning, and to establish and maintain predictable levels of donor/recipient chimerism. Methods Using stably transfected murine embryonic stem (ES) cells, we have generated a C57BL/6 transgenic mouse line with expression of MGMT(P140K) within the hematopoietic compartment for use as a standard source of donor HSC in such models. Functional characterisation of transgene expression was carried out in chemotherapy-treated transgenic mice and in allogeneic recipients of transgenic HSC. Results Expression of the transgene provided chemoprotection and allowed in vivo selection of MGMT(P140K)-expressing cells in transgenic mice after exposure to O6 -benzylguanine (BG) and N,N,-bis(2-chloroethyl)- N -nitrosourea (BCNU). In an allogeneic transplant experiment in which transgenic HSC were engrafted into 129 strain recipients following low intensity conditioning (Busulfan, anti-CD8, anti-CD40Ligand), MGMT(P140K)-expressing cells could be selected using chemotherapy. Conclusions This MGMT(P140K) transgenic mouse line provides a useful source of drug-selectable donor cells for the development of non-myeloablative allogeneic transplant models in which variation in transplant conditioning elements can be investigated independently of gene transfer efficiency. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Enhanced differentiation of embryonic stem cells using co-cultivation with hepatocytes

BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2008
Rebecca 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]

Supplementation-dependent differences in the rates of embryonic stem cell self-renewal, differentiation, and apoptosis

BIOTECHNOLOGY & BIOENGINEERING, Issue 5 2003
Sowmya 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]

Chemically Induced Cardiomyogenesis of Mouse Embryonic Stem Cells

CHEMBIOCHEM, Issue 2 2010
Albrecht Berkessel Prof. Dr.
Abstract A transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of ,-myosine heavy chain (,-MHC) promoter (p,-MHC-EGFP) was used to investigate the effects of (thio)urea and cinchona alkaloid derivatives on cardiomyogenesis. The screening of the compounds yielded cardiomyogenesis inducing substances with good (IV-5, V-4) to very good activities (II-16, IV-8), as determined by a 50 to 80,% increase in the EGFP fluorescence compared to untreated cells. Time-dependent screening approaches in which compounds were added at different developmental stages of the ES cells appeared to be of limited suitability for the identification of potential cellular targets. [source]