技术资料

Skin-derived precursor (SKP) cells are a valuable resource for tissue engineering and regenerative medicine,because they represent multipotent stem cells that differentiate into neural and mesodermal progenies. Previous studies suggest that the stem cell pool decreases with age. Here,we show that human multipotent SKP cells can be efficiently collected from adult cheek/chin skin,even in aged individuals of 70-78years. SKP cells were isolated from 38 skin samples by serum-free sphere culture and examined for the ability to differentiate into neural and mesodermal lineages. The number of spheres obtained from adult facial skin was significantly higher than that of trunk or extremity skin. SKP cells derived from cheek/chin skin exhibited a high ability to differentiate into neural and mesodermal cells relative to those derived from eyelid,trunk,or extremity skin. Furthermore,cheek/chin skin SKP cells were shown to express markers for undifferentiated stem cells,including a high expression level of the Sox9 gene. These results indicate that cheek/chin skin is useful for the recovery of multipotent stem cells for tissue engineering and regenerative therapy. View Publication

Nano- and microscale topographical cues play critical roles in the induction and maintenance of various cellular functions,including morphology,adhesion,gene regulation,and communication. Recent studies indicate that structure and function at the heart tissue level is exquisitely sensitive to mechanical cues at the nano-scale as well as at the microscale level. Although fabrication methods exist for generating topographical features for cell culture,current techniques,especially those with nanoscale resolution,are typically complex,prohibitively expensive,and not accessible to most biology laboratories. Here,we present a tunable culture platform comprised of biomimetic wrinkles that simulate the heart's complex anisotropic and multiscale architecture for facile and robust cardiac cell alignment. We demonstrate the cellular and subcellular alignment of both neonatal mouse cardiomyocytes as well as those derived from human embryonic stem cells. By mimicking the fibrillar network of the extracellular matrix,this system enables monitoring of protein localization in real time and therefore the high-resolution study of phenotypic and physiologic responses to in-vivo like topographical cues. View Publication

The ability to stimulate mammalian cells with light has significantly changed our understanding of electrically excitable tissues in health and disease,paving the way toward various novel therapeutic applications. Here,we demonstrate the potential of optogenetic control in cardiac cells using a hybrid experimental/computational technique. Experimentally,we introduced channelrhodopsin-2 into undifferentiated human embryonic stem cells via a lentiviral vector,and sorted and expanded the genetically engineered cells. Via directed differentiation,we created channelrhodopsin-expressing cardiomyocytes,which we subjected to optical stimulation. To quantify the impact of photostimulation,we assessed electrical,biochemical,and mechanical signals using patch-clamping,multielectrode array recordings,and video microscopy. Computationally,we introduced channelrhodopsin-2 into a classic autorhythmic cardiac cell model via an additional photocurrent governed by a light-sensitive gating variable. Upon optical stimulation,the channel opens and allows sodium ions to enter the cell,inducing a fast upstroke of the transmembrane potential. We calibrated the channelrhodopsin-expressing cell model using single action potential readings for different photostimulation amplitudes,pulse widths,and frequencies. To illustrate the potential of the proposed approach,we virtually injected channelrhodopsin-expressing cells into different locations of a human heart,and explored its activation sequences upon optical stimulation. Our experimentally calibrated computational toolbox allows us to virtually probe landscapes of process parameters,and identify optimal photostimulation sequences toward pacing hearts with light. ?? 2011 Biophysical Society. View Publication

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR,protein secreted from the liver aggregates and forms fibrils in target organs,chiefly the heart and peripheral nervous system,highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here,we describe detailed methodologies for the directed differentiation of protein folding disease-specific iPSCs into hepatocytes that produce mutant protein,and neural-lineage cells often targeted in disease. Methodologies are also described for the construction of multisystem models and drug screening using iPSCs. View Publication

Ex vivo expansion of hematopoietic stem cells (HSCs) is important for many clinical applications,and knowledge of the surface phenotype of ex vivo-expanded HSCs will be critical to their purification and analysis. Here,we developed a simple culture system for bone marrow (BM) HSCs using low levels of stem cell factor (SCF),thrombopoietin (TPO),insulin-like growth factor 2 (IGF-2),and fibroblast growth factor-1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses,there was a more than 20-fold increase in numbers of long-term (LT)-HSCs after a 10-day culture of total BM cells. Culture of BM side population" (SP) cells� View Publication

Human bone marrow (BM) or mobilized peripheral blood (mPB) CD34(+) cells have been shown to loose their stem cell quality during culture period more easily than those from cord blood (CB). We previously reported that human umbilical CB stem cells could effectively be expanded in the presence of human recombinant cytokines and a newly established murine bone marrow stromal cell line HESS-5. In this study we assessed the efficacy of this xenogeneic coculture system using human BM and mPB CD34(+) cells as materials. We measured the generation of CD34(+)CD38(-) cells and colony-forming units,and assessed severe-combined immunodeficient mouse-repopulating cell (SRC) activity using cells five days after serum-free cytokine-containing culture in the presence or the absence of a direct contact with HESS-5 cells. As compared with the stroma-free culture,the xenogeneic coculture was significantly superior on expansion of CD34(+)CD38(-) cells and colony-forming cells and on maintenance of SRC activity. The PKH26 study demonstrated that cell division was promoted faster in cells cocultured with HESS-5 cells than in cells cultured without HESS-5 cells. These results indicate that HESS-5 supports rapid generation of primitive progenitor cells (PPC) and maintains reconstituting ability of newly generated stem cells during ex vivo culture irrespective of the source of samples. This xenogeneic coculture system will be useful for ex vivo manipulation such as gene transduction to promote cell division and the generation of PPC and to prevent loss of stem cell quality. View Publication

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic $\$-cells. In other systems,Musashi genes regulate cell fate via Notch signaling,which we recently showed regulates $\$-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms,as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of $\$-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1,while it decreased Ins1 and Ins2 expression,revealing a molecular link between ER stress and $\$-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1,stimulate proliferation,inhibit apoptosis and reduce insulin expression,whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together,these results demonstrate overlapping,but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and $\$-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory $\$-cell proliferation and the loss of $\$-cell gene expression seen in specific phases of the progression to type 2 diabetes. View Publication

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here,we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein,decreased survival in longitudinal studies,and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening. View Publication

In up to one-third of patients with acute myeloid leukemia,a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM),and this is thought to function in cancer pathogenesis. Here,we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases,caspase-6 and -8,through direct interaction with their cleaved,active forms,but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia. View Publication

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways,including photoreceptor development,phototransduction,the retinoid cycle,cilia,and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants,two missense variants,and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1,which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383CtextgreaterT [p.Pro128Leu] and c.404TtextgreaterC [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore,CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP,including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore,our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy. View Publication

A battery of clonal assays for myeloid progenitor cells (HPP-CFC,CFU-gemm,CFU-gm,CFU-g) was utilized to evaluate the myelotoxicity of a series of alkylating agents representing the spectrum of clinical times to nadir. Bone marrow aspirates from normal volunteers were incubated with mechlorethamine,busulfan,melphalan,carmustine or lomustine for 1 h and then cultured in methylcellulose with 30% serum and cytokines. There was a concentration-dependent inhibition of colony formation and often a differential toxicity to the myeloid progenitors with the alkylators tested. On a molar basis,mechlorethamine and melphalan were the most toxic of the alkylator drugs to the myeloid precursors. The most sensitive progenitor was CFU-gemm with the lowest inhibitory concentration IC(70) concentrations for mechlorethamine,melphalan,carmustine and lomustine. Generally,there was great similarity for drug effects between CFU-g and CFU-gm with overlapping inhibition curves. HPP-CFC proved to be the least sensitive of the progenitors to the toxic actions of the drugs. While there was no correlation between the time to clinical neutropenic nadir and the most sensitive progenitor in the clonal assays,the CFU-gm assay remains a suitable method for determining the myelotoxic potential of cytotoxic agents. View Publication

The developmental potential of hematopoietic progenitors is restricted early on to either the erythromyeloid or lymphoid lineages. The broad developmental potential of Pax5(-/-) pro-B cells is in apparent conflict with such a strict separation,although these progenitors realize the myeloid and erythroid potential with lower efficiency compared to the lymphoid cell fates. Here we demonstrate that ectopic expression of the transcription factors C/EBPalpha,GATA1,GATA2 and GATA3 strongly promoted in vitro macrophage differentiation and myeloid colony formation of Pax5(-/-) pro-B cells. GATA2 and GATA3 expression also resulted in efficient engraftment and myeloid development of Pax5(-/-) pro-B cells in vivo. The myeloid transdifferentiation of Pax5(-/-) pro-B cells was accompanied by the rapid activation of myeloid genes and concomitant repression of B-lymphoid genes by C/EBPalpha and GATA factors. These data identify the Pax5(-/-) pro-B cells as lymphoid progenitors with a latent myeloid potential that can be efficiently activated by myeloid transcription factors. The same regulators were unable to induce a myeloid lineage switch in Pax5(+/+) pro-B cells,indicating that Pax5 dominates over myeloid transcription factors in B-lymphocytes. View Publication