搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

The ability to derive and stably maintain ground-state human pluripotent stem cells (hPSCs) that resemble the cells seen in vivo in the inner cell mass has the potential to be an invaluable tool for researchers developing stem cell-based therapies. To date,derivation of human naive-like pluripotent stem cell lines has been limited to a small number of lineages,and their long-term culturing remains problematic. We describe a protocol for genetic and phenotypic tagging,selecting and maintaining naive-like hPSCs. We tag hPSCs by GFP,expressed by the long terminal repeat (LTR7) of HERVH endogenous retrovirus. This simple and efficient protocol has been reproduced with multiple hPSC lines,including embryonic and induced pluripotent stem cells,and it takes ∼6 weeks. By using the reporter,homogeneous hPSC cultures can be derived,characterized and maintained for the long term by repeated re-sorting and re-plating steps. The HERVH-expressing cells have a similar,but nonidentical,expression pattern to other naive-like cells,suggesting that alternative pluripotent states might exist. View Publication

Encapsulation of donor islets using a hydrogel material is a well-studied strategy for islet transplantation,which protects donor islets from the host immune response. Replacement of donor islets by human embryonic stem cell (hESC) derived islets will also require a means of immune-isolating hESCs by encapsulation. However,a critical consideration of hESC differentiation is the effect of surrounding biophysical environment,in this case capsule biophysical properties,on differentiation. The objective of this study,thus,was to evaluate the effect of capsule properties on growth,viability,and differentiation of encapsulated hESCs throughout pancreatic induction. It was observed that even in the presence of soluble chemical cues for pancreatic induction,substrate properties can significantly modulate pancreatic differentiation,hence necessitating careful tuning of capsule properties. Capsules in the range of 4-7. kPa supported cell growth and viability,whereas capsules of higher stiffness suppressed cell growth. While an increase in capsule stiffness enhanced differentiation at the intermediate definitive endoderm (DE) stage,increased stiffness strongly suppressed pancreatic progenitor (PP) induction. Signaling pathway analysis indicated an increase in pSMAD/pAKT levels with substrate stiffness likely the cause of enhancement of DE differentiation. In contrast,sonic hedgehog inhibition was more efficient under softer gel conditions,which is necessary for successful PP differentiation. Statement of Significance: Cell replacement therapy for type 1 diabetes (T1D),affecting millions of people worldwide,requires the immunoisolation of insulin-producing islets by encapsulation with a semi-impermeable material. Due to the shortage of donor islets,human pluripotent stem cell (hPSC) derived islets are an attractive alternative. However,properties of the encapsulating substrate are known to influence hPSC cell fate. In this work,we determine the effect of substrate stiffness on growth and pancreatic fate of encapsulated hPSCs. We precisely identify the range of substrate properties conducive for pancreatic cell fate,and also the mechanism by which substrate properties modify the cell signaling pathways and hence cell fate. Such information will be critical in driving regenerative cell therapy for long term treatment of T1D. View Publication

Pericytes (PCs) are endothelium-associated cells that play an important role in normal vascular function and maintenance. We developed a method comparable to GMP quality protocols for deriving self-renewing perivascular progenitors from the human embryonic stem cell (hESC),line ESI-017. We identified a highly scalable,perivascular progenitor cell line that we termed PC-A,which expressed surface markers common to mesenchymal stromal cells. PC-A cells were not osteogenic or adipogenic under standard differentiation conditions and showed minimal angiogenic support function in vitro. PC-A cells were capable of further differentiation to perivascular progenitors with limited differentiation capacity,having osteogenic potential (PC-O) or angiogenic support function (PC-M),while lacking adipogenic potential. Importantly,PC-M cells expressed surface markers associated with pericytes. Moreover,PC-M cells had pericyte-like functionality being capable of co-localizing with human umbilical vein endothelial cells (HUVECs) and enhancing tube stability up to 6 days in vitro. We have thus identified a self-renewing perivascular progenitor cell line that lacks osteogenic,adipogenic and angiogenic potential but is capable of differentiation toward progenitor cell lines with either osteogenic potential or pericyte-like angiogenic function. The hESC-derived perivascular progenitors described here have potential applications in vascular research,drug development and cell therapy. View Publication

Organotin compounds,such as tributyltin (TBT),are well-known endocrine disruptors. TBT is also known to cause various forms of cytotoxicity,including neurotoxicity and immunotoxicity. However,TBT toxicity has not been identified in normal stem cells. In the present study,we examined the effects of TBT on cell growth in human induced pluripotent stem cells (iPSCs). We found that exposure to nanomolar concentrations of TBT decreased intracellular ATP levels and inhibited cell viability in iPSCs. Because TBT suppressed energy production,which is a critical function of the mitochondria,we further assessed the effects of TBT on mitochondrial dynamics. Staining with MitoTracker revealed that nanomolar concentrations of TBT induced mitochondrial fragmentation. TBT also reduced the expression of mitochondrial fusion protein mitofusin 1 (Mfn1),and this effect was abolished by knockdown of the E3 ubiquitin ligase membrane-associated RING-CH 5 (MARCH5),suggesting that nanomolar concentrations of TBT could induce mitochondrial dysfunction via MARCH5-mediated Mfn1 degradation in iPSCs. Thus,mitochondrial function in normal stem cells could be used to assess cytotoxicity associated with metal exposure. View Publication

Recent advances in developmental biology and stem cell technology have led to the engineering of functional organs in a dish. However,the limited size of these organoids and absence of a large circulatory system poses limits to its clinical translation. To overcome these issues,decellularized whole kidney scaffolds with native microstructure and extracellular matrix (ECM) are employed for kidney bioengineering,using human-induced pluripotent-stem-cell-derived renal progenitor cells and endothelial cells. To demonstrate ECM-guided cellular assembly,the present work is focused on generating the functional unit of the kidney,the glomerulus. In the repopulated organ,the presence of endothelial cells broadly upregulates the expression level of genes related to renal development. When the cellularized native scaffolds are implanted in SCID mice,glomeruli assembly can be achieved by co-culture of the renal progenitors and endothelial cells. These individual glomerular units are shown to be functional in the context of the whole organ using a simulated bio-reactor set-up with urea and creatinine excretion and albumin reabsorption. Our results indicate that the repopulation of decellularized native kidney using clinically relevant,expandable patient-specific renal progenitors and endothelial cells may be a viable approach for the generation of a functional whole kidney. View Publication

Chalcones are precursor compounds for flavonoid synthesis in plants,and they can also be synthesized in laboratory. Previous study has documented some of the pharmacological applications of these compounds. Estrogen has long been associated with the initiation and promotion of breast cancer. Inhibiting estrogen synthesis can be effective in the prevention and treatment of the disease. Since most breast cancers received estrogen supplied from local tissues,we employed a breast cancer cell line expressing aromatase to screen for the inhibitory potentials of five hydroxychalcones,i.e. 2-hydroxychalcone,2'-hydroxychalcone,4-hydroxychalcone,4,2',4'-trihydroxy-chalcone (isoquiritigenin),3,4,2',4'-tetrahydroxychalcone (butein). In the preliminary results,butein was found to be the strongest inhibitor among the tested compounds,and its IC(50) value was 3.75 microM. Subsequent enzyme kinetic study revealed that butein acted on aromatase with a mixed type of inhibition and the K(i) value was determined to be 0.32 microM. Cell proliferation assay indicated that the cell number increased by 10 nM-testosterone treatment was significantly reduced by 5 microM butein,and the administration of flutamide could not reverse the effect. The present study illustrated that butein was an aromatase inhibitor and a potential natural alternative for the chemoprevention or therapy of breast cancer. View Publication

The regulated production of neurons in the hippocampus throughout life underpins important brain functions such as learning and memory. Surprisingly,however,studies have so far failed to identify a resident hippocampal stem cell capable of providing the renewable source of these neurons. Here,we report that depolarizing levels of KCl produce a threefold increase in the number of neurospheres generated from the adult mouse hippocampus. Most interestingly,however,depolarizing levels of KCl led to the emergence of a small subpopulation of precursors (approximately eight per hippocampus) with the capacity to generate very large neurospheres (textgreater 250 microm in diameter). Many of these contained cells that displayed the cardinal properties of stem cells: multipotentiality and self-renewal. In contrast,the same conditions led to the opposite effect in the other main neurogenic region of the brain,the subventricular zone,in which neurosphere numbers decreased by approximately 40% in response to depolarizing levels of KCl. Most importantly,we also show that the latent hippocampal progenitor population can be activated in vivo in response to prolonged neural activity found in status epilepticus. This work provides the first direct evidence of a latent precursor and stem cell population in the adult hippocampus,which is able to be activated by neural activity. Because the latent population is also demonstrated to reside in the aged animal,defining the precise mechanisms that underlie its activation may provide a means to combat the cognitive deficits associated with a decline in neurogenesis. View Publication

OBJECTIVE: The T-cell receptor zeta (TCR zeta)-chain is a master sensor and regulator of lymphocyte responses. Loss of TCR zeta-chain expression has been documented during infectious and inflammatory diseases and defines a population of effector T cells (TCR zeta(dim) T cells) that migrate to inflamed tissues. We assessed the expression and functional correlates of circulating TCR zeta(dim) T cells in coronary artery disease. METHODS AND RESULTS: We examined the expression of TCR zeta-chain by flow cytometry in 140 subjects. Increased peripheral blood CD4(+) TCR zeta(dim) T cells were found in patients with acute coronary syndromes (ACS,n=66; median 5.3%,interquartile 2.6 to 9.1% of total CD4(+) T cells; Ptextless0.0001) compared to chronic stable angina (CSA,n=32; 1.6%; 1.0 to 4.1%) and controls (n=42; 1.5%; 0.5 to 2.9%). Such increase was significantly greater in ACS patients with elevated levels of C-reactive protein,and it persisted after the acute event. Moreover,TCR zeta(dim) cells were also more represented within CD8(+) T cell,NK,and CD4(+)CD28(null) T cell subsets in ACS compared to CSA and controls. Finally,CD4(+) and CD8(+) TCR zeta(dim) T cells isolated from ACS displayed an enhanced transendothelial migratory capacity. CONCLUSIONS: TCR zeta(dim) T cells,an effector T-cell subset with transendothelial migratory ability,are increased in ACS,and may be implicated in coronary instability. View Publication

The enumeration of circulating tumor cells has long been regarded as an attractive diagnostic tool,as circulating tumor cells are thought to reflect aggressiveness of the tumor and may assist in therapeutic decisions in patients with solid malignancies. However,implementation of this assay into clinical routine has been cumbersome,as a validated test was not available until recently. Circulating tumor cells are rare events which can be detected specifically only by using a combination of surface and intracellular markers,and only recently a number of technical advances have made their reliable detection possible. Most of these new techniques rely on a combination of an enrichment and a detection step. This review addresses the assays that have been described so far in the literature,including the enrichment and detection steps and the markers used in these assays. We have focused on breast cancer as most clinical studies on CTC detection so far have been done in these patients. View Publication