Cellular Reprogramming of Human Peripheral Blood Cells

Breakthroughs in cell fate conversion have made it possible to generate large quantities of patient-specific cells for regenerative medicine. Due to multiple advantages of peripheral blood cells over fibroblasts from skin biopsy, the use of blood mononuclear cells （MNCs） instead of skin fibroblasts will expedite reprogramming research and broaden the application of reprogramming technology. This review discusses current progress and challenges of generating induced pluripotent stem cells （iPSCs） from peripheral blood MNCs and of in vitro and in vivo conversion of blood cells into cells of therapeutic value, such as mesenchymal stem cells, neural cells and hepatocytes. An optimized design of lentiviral vectors is necessary to achieve high reprogramming efficiency of peripheral blood cells. More recently, non-integrating vectors such as Sendai virus and episomal vectors have been successfully employed in generating integration-free iPSCs and somatic stem cells.     

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.     

Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures

To identify accessible and permissive human cell types for efficient derivation of induced pluripotent stem cells (iPSCs), we investigated epigenetic and gene expression signatures of multiple postnatal cell types such as fibroblasts and blood cells. Our analysis suggested that newborn cord blood (CB) and adult peripheral blood (PB) mononuclear cells (MNCs) display unique signatures that are closer to iPSCs and human embryonic stem cells (ESCs) than age-matched fibroblasts to iPSCs/ESCs, thus making blood MNCs an attractive cell choice for the generation of integration-free iPSCs. Using an improved EBNA1/OriP plasmid expressing 5 reprogramming factors, we demonstrated highly efficient reprogramming of briefly cultured blood MNCs. Within 14 days of one-time transfection by one plasmid, up to 1000 iPSC-like colonies per 2 million transfected CB MNCs were generated. The efficiency of deriving iPSCs from adult PB MNCs was approximately 50-fold lower, but could be enhanced by inclusion of a second EBNA1/OriP plasmid for transient expression of additional genes such as SV40 T antigen. The duration of obtaining bona fide iPSC colonies from adult PB MNCs was reduced to half (～14 days) as compared to adult fibroblastic cells (28-30 days). More than 9 human iPSC lines derived from PB or CB blood cells are extensively characterized, including those from PB MNCs of an adult patient with sickle cell disease. They lack V(D)J DNA rearrangements and vector DNA after expansion for 10-12 passages. This facile method of generating integration-free human iPSCs from blood MNCs will accelerate their use in both research and future clinical applications.     

Derivation of male germ cells from induced pluripotent stem cells by inducers: A review

Induced pluripotent stem cells (iPSCs) refer to stem cells that are artificially produced using a new technology known as cellular reprogramming, which can use gene transduction in somatic cells. There are numerous potential applications for iPSCs in the field of stem cell biology becauase they are able to give rise to several different cell features of lineages such as three-germ layers. Primordial germ cells, generated via in vitro differentiation of iPSCs, have been demonstrated to produce functional gametes. Therefore, in this review we discussed past and recent advances in the in vitro differentiation of germ cells using pluripotent stem cells with an emphasis on iPSCs. Although this domain of research is still in its infancy, exploring development mechanisms of germ cells is promising, especially in humans, to promote future reproductive and developmental engineering technologies. While few studies have evaluated the ability and efficiency of iPSCs to differentiate toward male germ cells in vitro by different inducers, the given effect was investigated in this review.     

Transfection,Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.     

诱导多潜能干细胞(iPSCs)的研究与应用进展

诱导多潜能干细胞(induced pluripotent stem cells,iPSCs)是体细胞在外源因子作用下,经直接细胞核程序重整而重新获得多潜能的干细胞.iPSCs在疾病的模型建立与机理研究、细胞治疗、药物的发现与评价等方面有着巨大的潜在应用价值.在过去几年中,科学家们致力于改进体细胞重编程技术并取得许多突破.然而,为实现其在临床上的应用,必须克服体细胞重编程效率低和iPSCs成瘤风险两大挑战,而且重编程机制有待进一步阐明.结合iPSCs最新研究成果,评述了有关领域国内外研究进展,重点讨论当前存在问题,并展望未来研究方向.     

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy.     

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)¹. While improvements in several gene delivery methods have been described^2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor¹¹, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.     

Efficient feeder-free episomal reprogramming with small molecules

Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint-free), and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3β inhibitor CHIR99021, TGF-β/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs.