This article discusses the implications of the recent discovery that rat bone marrow-derived multipotent adult progenitor cells (rMAPCs), a cell type with broad somatic differentiation potential but of uncertain lineage identity, are similar to rat blastocyst-derived extraembryonic endoderm precursor (rXENP) cells, which appear to represent the committed extraembryonic endoderm precursor of the blastocyst. It was found that under rMAPC culture conditions, rXENP cells can be homogeneously cultured and similar cells, named rat hypoblast stem cells (rHypoSCs), can be derived from rat blastocysts more rapidly and directly. The detailed comparison of rHypoSCs, rXENP cells, and rMAPCs revealed highly similar gene expression profiles and developmental potentials. The significance of these findings for embryology, stem cell biology, and medicine is discussed. Specifically, the results assign a lineage identity to rMAPCs, indicate that rMAPCs originated by environmental reprogramming, and imply that HypoSCs, XENP cells, and MAPCs possess lineage plasticity. The MAPC-HypoSC relation also strengthens the consistency of rat and mouse embryology and consequently the idea that HypoSCs represent the committed extraembryonic endoderm precursor of the blastocyst. On this basis, it is argued that the direct comparison of HypoSCs (now available in pure form) with embryonic stem cells will be highly useful for the understanding of pluripotency and plasticity. Finally, the new findings suggest an explanation for an obscure observation on stem cell-induced transplantation tolerance. Thus, the HypoSC/XENP/MAPC phenotype provides a unique but broadly instructive model with which to study stem cell plasticity, reprogramming, and transplantation tolerance, all central themes in regenerative medicine.
Adenylate kinase isozyme 4 (AK4) belongs to a family of nucleotide monophosphate kinases involved in energy metabolism. Recently, AK4 was reported to play a role in protection from stress: In HEK293 cells, hypoxia increases AK4 expression but does not affect proliferation or viability, while RNA interference (RNAi) directed against AK4 inhibits proliferation and promotes death. By contrast, we show here that HepG2 cells showed much higher AK4 levels, which decreased under hypoxia along with markedly reduced cell proliferation and increased cell death. Nevertheless, RNAi directed against AK4 inhibited cell proliferation and caused death in both cell types, although cell cycle parameters were affected only in HepG2 cells. Hence reductions of AK4 levels were always associated with cell death. These results extend the notion of a stress-protective function of AK4 to a novel physiological context and show that AK4-mediated stress protection is not limited to one particular death scenario. Our data also allow the hypothesis that the different basal AK4 levels reflect different basal stress levels, causing alternative responses to additional stress.
?-cell replacement may efficiently cure type 1 diabetic (T1D) patients whose insulin-secreting ?-cells have been selectively destroyed by autoantigen-reactive T cells. To generate insulin-secreting cells we used two cell sources: rat multipotent adult progenitor cells (rMAPC) and the highly similar rat extra-embryonic endoderm precursor (rXEN-P) cells isolated under rMAPC conditions from blastocysts (rHypoSC). rMAPC/rHypoSC were sequentially committed to definitive endoderm, pancreatic endoderm, and ?-cell like cells. On day 21, 20% of rMAPC/rHypoSC progeny expressed Pdx1 and C-peptide. rMAPCr/HypoSC progeny secreted C-peptide under the stimulus of insulin agonist carbachol, and was inhibited by the L-type voltage-dependent calcium channel blocker nifedipine. When rMAPC or rHypoSC differentiated d21 progeny were grafted under the kidney capsule of streptozotocin-induced diabetic nude mice, hyperglycemia reversed after 4 weeks in 6/10 rMAPC- and 5/10 rHypoSC-transplanted mice. Hyperglycemia recurred within 24 hours of graft removal and the histological analysis of the retrieved grafts revealed presence of Pdx1-, Nkx6.1- and C-peptide-positive cells. The ability of both rMAPC and HypoSC to differentiate to functional ?-cell like cells may serve to gain insight into signals that govern ?-cell differentiation and aid in developing culture systems to commit other (pluripotent) stem cells to clinically useful ?-cells for cell therapy of T1D.
It has long been known that mammalian enterocytes coexpress two members of the fatty acid-binding protein (FABP) family, the intestinal FABP (IFABP) and the liver FABP (LFABP). Both bind long-chain fatty acids and have similar though not identical distributions in the intestinal tract. While a number of in vitro properties suggest the potential for different functions, the underlying reasons for expression of both proteins in the same cells are not known. Utilizing mice genetically lacking either IFABP or LFABP, we directly demonstrate that each of the enterocyte FABPs participates in specific pathways of intestinal lipid metabolism. In particular, LFABP appears to target fatty acids toward oxidative pathways and dietary monoacylglycerols toward anabolic pathways, while IFABP targets dietary fatty acids toward triacylglycerol synthesis. The two FABP-null models also displayed differences in whole body response to fasting, with LFABP-null animals losing less fat-free mass and IFABP-null animals losing more fat mass relative to wild-type mice. The metabolic changes observed in both null models appear to occur by nontranscriptional mechanisms, supporting the hypothesis that the enterocyte FABPs are specifically trafficking their ligands to their respective metabolic fates.
Fatty acid binding proteins (FABPs) are essential for energy production and long-chain polyunsaturated fatty acid-related signaling in the brain and other tissues. Of various FABPs, heart-type fatty acid binding protein (H-FABP, FABP3) is highly expressed in neurons of mature brain and plays a role in arachidonic acid incorporation into brain and heart cells. However, the precise function of H-FABP in brain remains unclear. We previously demonstrated that H-FABP is associated with the dopamine D(2) receptor long isoform (D2LR) in vitro. Here, we confirm that H-FABP binds to dopamine D(2) receptor (D2R) in brain extracts and colocalizes immunohistochemically with D2R in the dorsal striatum. We show that H-FABP is highly expressed in acetylcholinergic interneurons and terminals of glutamatergic neurons in the dorsal striatum of mouse brain but absent in dopamine neuron terminals and spines in the same region. H-FABP knock-out (KO) mice showed lower responsiveness to methamphetamine-induced sensitization and enhanced haloperidol-induced catalepsy compared with wild-type mice, indicative of D2R dysfunction. Consistent with the latter, aberrant increased acetylcholine (ACh) release and depolarization-induced glutamate (Glu) release were observed in the dorsal striatum of H-FABP KO mice. Furthermore, phosphorylation of CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) and ERK (extracellular signal-regulated kinase) was significantly increased in the dorsal striatum. We confirmed elevated ERK phosphorylation following quinpirole-mediated D2R stimulation in H-FABP-overexpressing SHSY-5Y human neuroblastoma cells. Together, H-FABP is highly expressed in ACh interneurons and glutamatergic terminals, thereby regulating dopamine D2R function in the striatum.
Previous attempts to isolate pluripotent cell lines from rat preimplantation embryo in mouse embryonic stem (ES) cell culture conditions (serum and LIF) were unsuccessful, however the resulting cells exhibited the expression of such traditional pluripotency markers as SSEA-1 and alkaline phosphatase. We addressed the question, which kind of cell lineages are produced from rat preimplantation embryo under "classical" mouse ES conditions.
We have previously found that certain stem cells that are derived from rat blastocysts and named extraembryonic endoderm precursor (XEN-P) cells show a unique molecular signature sharing some of the characteristics of embryonic stem cells (ES), trophoblast stem cells (TS), and extraembryonic endoderm stem cells (XEN). These XEN-P cells are positive for AP, SSEA1, Oct4, and Rex1 markers similar to ES cells and also express signature markers of TS-eomesodermin (Eomes) and XEN-Gata6. Here we show that these cells integrate into the visceral and parietal extraembryonic endoderm lineages as well as into the inner cell mass (ICM), the primitive endoderm, and the polar and mural trophectoderm (TE) of cultured embryos. In addition, we find that the XEN-P cells colonize yolk sac and contribute to trophoblast lineages of postimplantation embryos following transfer to surrogate mothers. We also find that the XEN-P cell culture propagates by shedding cell clusters into the media in addition to typical expansion of colonies. Interestingly, the cell cultures exist as mixed populations of two interconvertible phenotypes of flat and round cells with preferential expression of stem cell markers Oct4 and SSEA1 in round cells. We believe these cells represent a metastable stage during ICM cellular segregation. These results are important for developing hypotheses of cell fate plasticity in the ICM and provide a model for the study of development and differentiation along the extraembryonic lineages.
In view of the well-known phenomenon of trophoblast immune privilege, trophoblast stem cells (TSCs) might be expected to be immune privileged, which could be of interest for cell or gene therapies. Yet in the ectopic sites tested so far, TSC transplants fail to show noticeable immune privilege and seem to lack physiological support. However, we show here that after portal venous injection, green fluorescent protein (GFP)-labeled TSCs survive for several months in the livers of allogeneic female but not male mice. Gonadectomy experiments revealed that this survival does not require the presence of ovarian hormones but does require the absence of testicular factors. By contrast, GFP-labeled allogeneic embryonic stem cells (ESCs) are reliably rejected; however, these same ESCs survive when mixed with unlabeled TSCs. The protective effect does not require immunological compatibility between ESCs and TSCs. Tumors were not observed in animals with either successfully engrafted TSCs or coinjected ESCs. We conclude that in a suitable hormonal context and location, ectopic TSCs can exhibit and confer immune privilege. These findings suggest applications in cell and gene therapy as well as a new model for studying trophoblast immunology and physiology.
Our group has synthesized technetium-labeled fatty acids (FA) that are extracted into the myocardium and sequestered due to heart-type fatty acid binding protein (H-FABP) binding. In this article, we further address the detailed subcellular distribution and potential myocardial metabolism of [(99m)Tc]"4+1" FA.
(13)C, (18)F and (123)I fatty acids (FA) are used for myocardial imaging. Recently, our group showed that [(99m)Tc]-labeled "4+1" FA are extracted into the rat and guinea pig myocardium. The present study evaluates determinants of myocardial uptake and whole body biodistribution of these FA derivatives.
The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4.
We recently isolated hypoblast stem cells (HypoSC), which are related to embryonic stem (ES) cells. ES cells efficiently perform homologous recombination (HR) and lack X chromosome inactivation (Xi), but it is unknown whether the same applies to HypoSC. Using the X-linked hypoxanthine phosphoribosyl transferase (HPRT) gene, we find that HypoSC perform HR with similar frequency as ES cells. Monoallelic targeting in female HypoSC eliminated HPRT gene expression, implying epigenetic inactivation of the other allele. Although density-induced differentiation complicated selection, the targeted clones maintained their original properties. These results will facilitate targeted genetic manipulation of HypoSC and the study of Xi.
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