参考文献

A defined xeno-free system for patient-specific iPSC derivation and differentiation is required for translation to clinical applications. However,standard somatic cell reprogramming protocols rely on using MEFs and xenogeneic medium,imposing a significant obstacle to clinical translation. Here,we describe a well-defined culture system based on xeno-free media and LN521 substrate which supported i) efficient reprogramming of normal or diseased skin fibroblasts from human of different ages into hiPSCs with a 15-30 fold increase in efficiency over conventional viral vector-based method; ii) long-term self-renewal of hiPSCs; and iii) direct hiPSC lineage-specific differentiation. Using an excisable polycistronic vector and optimized culture conditions,we achieved up to 0.15%-0.3% reprogramming efficiencies. Subsequently,transgene-free hiPSCs were obtained by Cre-mediated excision of the reprogramming factors. The derived iPSCs maintained long-term self-renewal,normal karyotype and pluripotency,as demonstrated by the expression of stem cell markers and ability to form derivatives of three germ layers both in vitro and in vivo. Importantly,we demonstrated that Parkinson's patient transgene-free iPSCs derived using the same system could be directed towards differentiation into dopaminergic neurons under xeno-free culture conditions. Our approach provides a safe and robust platform for the generation of patient-specific iPSCs and derivatives for clinical and translational applications. textcopyright 2013 Elsevier Ltd. View Publication

Here we provide a protocol for the directed differentiation of hEPI-NCSC into midbrain dopaminergic neurons,which degenerate in Parkinson's disease. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. The experimental design is distinctly different from conventional protocols for embryonic stem cells and induced pluripotent stem (iPS) cells. It includes pre-differentiation of the multipotent hEPI-NCSC into neural stem cell-like cells,followed by ventralizing,patterning,continued exposure to the TGFβ receptor inhibitor,SB431542,and at later stages of differentiation the presence of the WNT inhibitor,IWP-4. All cells expressed A9 midbrain dopaminergic neuron progenitor markers with gene expression levels comparable to those in normal human substantia nigra. The current study shows for the first time that virtually homogeneous populations of dopaminergic neurons can be derived ex vivo from somatic stem cells without the need for purification,with useful timeliness and high efficacy. This novel development is an important first step towards the establishment of fully functional dopaminergic neurons from an ontologically relevant stem cell type,hEPI-NCSC. View Publication

There is a great deal of uncertainty on how low (≤ 0.1 Gy) doses of ionizing radiation (IR) affect human cells,partly due to a lack of suitable experimental model systems for such studies. The uncertainties arising from low-dose IR human data undermine practical societal needs to predict health risks emerging from diagnostic medical tests' radiation,natural background radiation,and environmental radiological accidents. To eliminate a variability associated with remarkable differences in radioresponses of hundreds of differentiated cell types,we established a novel,human embryonic stem cell (hESC)-based model to examine the radiobiological effects in human cells. Our aim is to comprehensively elucidate the gene expression changes in a panel of various hESC lines following low IR doses of 0.01; 0.05; 0.1 Gy; and,as a reference,relatively high dose of 1 Gy of IR. Here,we examined the dynamics of transcriptional changes of well-established IR-responsive set of genes,including CDKN1A,GADD45A,etc. at 2 and 16 h post-IR,representing early" and "late" radioresponses of hESCs. Our findings suggest the temporal- and hESC line-dependence of stress gene radioresponses with no statistically significant evidence for a linear dose-response relationship within the lowest doses of IR exposures." View Publication

Induced pluripotent stem cells (iPSCs) derived from patients with neurodegenerative disease generally lack neuropathological confirmation,the gold standard for disease classification and grading of severity. The use of tissue with a definitive neuropathological diagnosis would be an ideal source for iPSCs. The challenge to this approach is that the majority of biobanked brain tissue was not meant for growing live cells,and thus was not frozen in the presence of cryoprotectants such as DMSO. PMID: 24398250 View Publication

Induced pluripotent stem cells (iPS) can differentiate into cardiomyocytes (CM) and represent a promising form of cellular therapy for heart regeneration. However,residual undifferentiated iPS derivates (iPSD),which are not fully eliminated by cell differentiation or purification protocols,may form tumors after transplantation,thus compromising therapeutic application. Inhibition of stearoyl-coA desaturase (SCD) has recently been reported to eliminate undifferentiated human embryonic stem cells,which share many features with iPSD. Here,we tested the effects of PluriSin1,a small-molecule inhibitor of SCD,on iPS-derived CM. We found that plurisin1 treatment significantly decreased the mRNA and protein level of Nanog,a marker for both cell pluripotency and tumor progression; importantly,we provide evidence that PluriSin1 treatment at 20 µM for 1 day significantly induces the apoptosis of Nanog-positive iPSD. In addition,PluriSin1 treatment at 20 µM for 4 days diminished Nanog-positive stem cells in cultured iPSD while not increasing apoptosis of iPS-derived CM. To investigate whether PluriSin1 treatment prevents tumorigenicity of iPSD after cell transplantation,we intramyocardially injected PluriSin1- or DMSO-treated iPSD in a mouse model of myocardial infarction (MI). DMSO-treated iPSD readily formed Nanog-expressing tumors 2 weeks after injection,which was prevented by treatment with PluriSin1. Moreover,treatment with PluriSin1 did not change the expression of cTnI,α-MHC,or MLC-2v,markers of cardiac differentiation (Ptextgreater0.05,n = 4). Importantly,pluriSin1-treated iPS-derived CM exhibited the ability to engraft and survive in the infarcted myocardium. We conclude that inhibition of SCD holds the potential to enhance the safety of therapeutic application of iPS cells for heart regeneration. View Publication

Structural bone allografts are widely used in the clinic to treat critical sized bone defects,despite lacking the osteoinductive characteristics of live autografts. To address this,we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets,which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates,as a tissue-engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets,suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo,allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of X-ray plain radiography,3D microCT,histology,and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation,as well as graft-host osteointegration. Moreover,a large periosteal callus was observed spanning the allografts with MSC sheets,which partially mimics live autograft healing. Finally,biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together,MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair,demonstrating the feasibility of this tissue engineering solution for massive allograft healing. View Publication

Induced pluripotent stem cells (iPSCs) are becoming mainstream tools to study mechanisms of development and disease. They have a broad range of applications in understanding disease processes,in vitro testing of novel therapies,and potential utility in regenerative medicine. Although the techniques for generating iPSCs are becoming more straightforward,scientists can expend considerable resources and time to establish this technology. A major hurdle is the accurate determination of valid iPSC-like colonies that can be selected for further cloning and characterization. In this study,we describe the use of a gammaretroviral vector encoding a fluorescent marker,mRFP1,to not only monitor the efficiency of initial transduction but also to identify putative iPSC colonies through silencing of mRFP1 gene as a consequence of successful reprogramming. View Publication

BACKGROUND Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC),particularly the development of oropharyngeal squamous cell carcinoma (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies,and it is postulated that they are responsible for disease recurrence and/or progression. Because the prognoses of patients with HPV16-positive and HPV-negative HNSCC are distinct,the authors sought to determine whether differences in the number of CSCs could account for this clinical observation. METHODS CSC populations in HPV16-positive and HPV-negative HNSCC were assessed using a proprietary assay based on expression of the enzyme aldehyde dehydrogenase (ALDH),an in vitro tumorsphere formation assay,and an in vivo limiting cell dilution in nonobese diabetic/severe combined immunodeficiency mice. A high-density tissue microarray was stained with ALDH1,a CSC marker,to determine the association between CSCs and HPV16-positive/HPV-negative OPSCC. RESULTS HPV16-positive HNSCC had a greater intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 has been identified as a major mechanism for the elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using tumors from patients with HPV16-positive and HPV-negative OPSCC indicated that the CSC frequency was 62.5-fold greater in an HPV16-positive OPSCC tumor than in an HPV-negative OPSCC tumor. Primary tumors from patients with HPV16-positive OPSCC were associated with elevated tumor ALDH1 staining,further extending the association between HPV16 and CSCs. CONCLUSIONS The current data and the clinical observation that patients with HPV16-positive HNSCC respond more favorably to current treatment paradigms than patients with HPV-negative HNSCC support the suggestion that CSC phenotype is not homogeneous. Therefore,the reliance on the CSC number may be insufficient to accurately assess the potential of a particular tumor for disease recurrence and/or progression. View Publication

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4),encoding spastin,are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons,we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684CtextgreaterT nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased,which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin,these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein,p60 katanin,may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length,branching,numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore,our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients. View Publication

Recurrent chromosomal alterations have been repeatedly reported in cultured human embryonic stem cells (hESCs). The effects of these alterations on the capability of pluripotent cells to differentiate and on growth potential of their specific differentiated derivatives remain unclear. Here,we report that the hESC lines HUES-7 and -9 carrying multiple chromosomal alterations produce in vitro mesenchymal stem cells (MSCs) that show progressive growth arrest and enter senescence after 15 and 16 passages,respectively. There was no difference in their proliferative potential when compared with bone marrow-derived MSCs. Array comparative genomic hybridization analysis (aCGH) of hESCs and their mesenchymal derivatives revealed no significant differences in chromosomal alterations,suggesting that genetically altered hESCs are not selected out during differentiation. Our findings indicate that genetically unstable hESCs maintain their capacity to differentiate in vitro into MSCs,which exhibit an in vitro growth pattern of normal MSCs and not that of transformed cells. View Publication