体细胞重编程为诱导多能干细胞

诱导多功能性干细胞(induced pluripotent stem cells,iPS细胞)是通过导入特定的转录因子(如Oct3/4、Sox2、c-Myc和Klf4等)将体细胞诱导重编程为多能性干细胞,其功能与胚胎干细胞相似.iPS细胞的建立,在生命科学领域引起了新的轰动.目前,iPS细胞的研究领域在转录因子的优化、iPS细胞的筛选、载体的运用、体细胞种类的选择和iPS细胞的应用等方面取得突破进展,但仍然存在致癌性、效率低等一系列急需解决的问题.     

Pluripotent Human Stem Cells

The need for new and improved pharmacotherapies in medicine, high late-stage compound attrition in drug discovery, and upcoming patent expirations is driving interest by the pharmaceutical industry in pluripotent stem cells for in vitro modeling and early-stage testing of toxicity and target engagement. In particular, human embryonic and induced pluripotent stem cells represent potentially cost-effective and accessible sources of organ-specific cells that foretell in vivo human tissue response to new chemical entities. Here we consider the potential of these cells as novel tools for drug development, including toxicity screening and metabolic profiling. We hold that despite various challenges to translating proof-of-concept screening platforms to industrial use, the promise of research is considerable, and close to being realized.     

Pluripotent Stem Cells as a Robust Source of Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) have been extensively studied over the past years for the treatment of different diseases. Most of the ongoing clinical trials currently involve the use of MSC derived from adult tissues. This source may have some limitations, particularly with therapies that may require extensive and repetitive cell dosage. However, nowadays, there is a staggering growth in literature on a new source of MSC. There is now increasing evidence about the mesenchymal differentiation from pluripotent stem cell (PSC). Here, we summarize the current knowledge of pluripotent-derived mesenchymal stem cells (PD-MSC). We present a historical perspective on the subject, and then discuss some critical questions that remain unanswered.     

诱导多潜能干细胞

通过病毒载体导入4个外源转录因子Oct4、Sox2、c-Myc、Klf或者Oct4、Sox2、Nanog、Lin28入体细胞,可以诱导产生具有胚胎干细胞特性相似的诱导多潜能干细胞(induced pluripotent stem cells,iPS).iPS在疾病治疗和药物研究等领域具有非常重要的应用前景,但是目前存在诱导效率低以及致肿瘤性等缺点,采用改良方法诱导产生iPS是将来研究的重点.     

Induced Pluripotent Stem Cells from Human Hair Follicle Mesenchymal Stem Cells

Reprogramming of somatic cells into inducible pluripotent stem cells (iPSCs) provides an alternative to using embryonic stem cells (ESCs). Mesenchymal stem cells derived from human hair follicles (hHF-MSCs) are easily accessible, reproducible by direct plucking of human hairs. Whether these hHF-MSCs can be reprogrammed has not been previously reported. Here we report the generation of iPSCs from hHF-MSCs obtained by plucking several hairs. hHF-MSCs were isolated from hair follicle tissues and their mesenchymal nature confirmed by detecting cell surface antigens and multilineage differentiation potential towards adipocytes and osteoblasts. They were then reprogrammed into iPSCs by lentiviral transduction with Oct4, Sox2, c-Myc and Klf4. hHF-MSC-derived iPSCs appeared indistinguishable from human embryonic stem cells (hESCs) in colony morphology, expression of alkaline phosphotase, and expression of specific hESCs surface markers, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, Nanog, Oct4, E-Cadherin and endogenous pluripotent genes. When injected into immunocompromised mice, hHF-MSC-derived iPSCs formed teratomas containing representatives of all three germ layers. This is the first study to report reprogramming of hHF-MSCs into iPSCs.     

Induced Pluripotent Stem Cells: Fundamentals and Applications of the Reprogramming Process and its Ramifications on Regenerative Medicine

Objective  

To provide a comprehensive source of information about the reprogramming process and induced pluripotency.     

Promising New Sources for Pluripotent Stem Cells

Recent findings have placed stem cell research at the forefront of biomedical sciences. Basic research on embryonic stem cells (ESCs) has contributed to our knowledge about the developmental potential and plasticity of stem cells. Furthermore, it has raised hope to use these cells as potential source for regenerative medicine and tissue replacement after injury or disease. Unfortunately, ESCs can also form tumors and they are ethically controversial because they originate from human embryos. This review summarizes findings and therapeutic applications of ESCs and their alternatives: adult stem cells and iPS cells.     

Effective Differentiation of Induced Pluripotent Stem Cells Into Dental Cells

Background: A biotooth is defined as a complete living tooth, made in laboratory cultures from a spontaneous interplay between epithelial and mesenchymal cell-based frontal systems. A good solution to these problems is to use induced pluripotent stem cells (iPSCs). However, no one has yet formulated culture conditions that effectively differentiate iPSCs into dental epithelial and dental mesenchymal cells phenotypes analogous to those present in tooth development. Results: Here, we tried to induce differentiation methods for dental epithelial cells (DEC) and dental mesenchymal cells from iPSCs. For the DEC differentiation, the conditional media of SF2 DEC was adjusted to embryoid body. Moreover, we now report on a new cultivation protocol, supported by transwell membrane cell culture that make it possible to differentiate iPSCs into dental epithelial and mesenchymal cells with abilities to initiate the first stages in de novo tooth formation. Conclusions: Implementation of technical modifications to the protocol that maximize the number and rate of iPSC differentiation, into mesenchymal and epithelial cell layers, will be the next step toward growing an anatomically accurate biomimetic tooth organ. Developmental Dynamics 248:129–139, 2019. © 2018 Wiley Periodicals, Inc.     

Stem Cells and Muscle Diseases

In the past recent years, basic science work and initial clinical trials have provided starting evidence that stem cells are of potential value for treatment of certain human diseases, where they could help to regenerate tissues which are defective because of either genetic or acquired diseases. This area represents an emerging field of biomedicine based on a series of new discoveries in the field of stem cell biology and developmental biology that have made possible to isolate and expand stem cells from many human tissues. Additional evidence has also revealed the role of tissue environment that, by releasing a complex mixture of cytokines and growth factors, can influence the recruitment and functional integration of stem cells into specific organs. However, there is an urgent need for more advancement in basic biology of stem cells and related topics, which will be instrumental for the implementation of stem cell-based therapy at the clinical level, as treatment accessibility will depend on the acquisition of sufficient knowledge to develop adequate technologies to produce sufficient cell numbers and to drive their differentiating potentials.     

Electrically Guiding Migration of Human Induced Pluripotent Stem Cells

A major road-block in stem cell therapy is the poor homing and integration of transplanted stem cells with the targeted host tissue. Human induced pluripotent stem (hiPS) cells are considered an excellent alternative to embryonic stem (ES) cells and we tested the feasibility of using small, physiological electric fields (EFs) to guide hiPS cells to their target. Applied EFs stimulated and guided migration of cultured hiPS cells toward the anode, with a stimulation threshold of <30 mV/mm; in three-dimensional (3D) culture hiPS cells remained stationary, whereas in an applied EF they migrated directionally. This is of significance as the therapeutic use of hiPS cells occurs in a 3D environment. EF exposure did not alter expression of the pluripotency markers SSEA-4 and Oct-4 in hiPS cells. We compared EF-directed migration (galvanotaxis) of hiPS cells and hES cells and found that hiPS cells showed greater sensitivity and directedness than those of hES cells in an EF, while hES cells migrated toward cathode. Rho-kinase (ROCK) inhibition, a method to aid expansion and survival of stem cells, significantly increased the motility, but reduced directionality of iPS cells in an EF by 70–80%. Thus, our study has revealed that physiological EF is an effective guidance cue for the migration of hiPS cells in either 2D or 3D environments and that will occur in a ROCK-dependent manner. Our current finding may lead to techniques for applying EFs in vivo to guide migration of transplanted stem cells.     

Redefining the Concept of Standardization for Pluripotent Stem Cells

In this report we review the concept of standardization and propose an exhaustive framework for the proper management of technology on pluripotent stem cells based on studies of global and regional initiatives. We demonstrated detailed observational analysis on global initiatives for the standardization of related technologies as well as regional attempts with particular interest in the cases of the UK, the US and Japan. Consequently, we came up two fundamental issues: first, these initiatives and attempts tend to be limited to each of currently existing categories of pluripotent stem cells, whereas the technological opportunity to enable clinical/commercial application is equally open to all stem cell types. Second, the subject to be examined for standardization is set to a quite narrow range compared to precedent practices in other industrial sectors. To address these issues, we propose a strategic framework for standardization with an emphasis on comprehensiveness covering various technological opportunities and consistency to learning in the management science. By utilizing this framework development of intellectual property rights not only through patents but through taking leadership in standardization can be considered as means for improving research and development competence. Of particular concerns is the formation of quality standards for final products/services and core elemental technology, especially specific pluripotent stem cell lines. Furthermore, we inferred two stages of the standardization process, individualization where a particular product/service is qualified by the market, then standardization where the elemental technology is subsequently established as compatibility standards.     

Pluripotent Stem Cells: Origin, Maintenance and Induction

Pluripotency is defined as the potential of a cell to differentiate into cells of the three germ layers: endoderm, mesoderm and ectoderm. In vivo, the presence of pluripotent stem cells is transient during the very early embryo. However, immortal cell lines with the same properties can be obtained in vitro and grown indefinitely in laboratories under specific conditions. These cells can be induced to differentiate into all the cell types of the organism through different assays, thereby proving their functional pluripotency. This review focuses on the pluripotent stem cells of mammals, giving special attention to the comparison between mouse and human. In particular, embryonic stem cells, epiblast-derived stem cells, primordial germ cells, embryonic germ cells, very small embryonic-like cells and induced pluripotent stem cells will be compared in terms of the following: in vivo specification and location; surface and intracellular markers; in vitro dependence on growth factors; signal transduction pathways; epigenetic characteristics; and pluripotency genes and functional assays.     

Laser-Assisted Photoablation of Human Pluripotent Stem Cells from Differentiating Cultures

Due to their pluripotency and their self-renewal capacity, human pluripotent stem cells (hPSC) provide fascinating perspectives for biomedical applications. In the long term, hPSC-derived tissue-specific cells will constitute an important source for cell replacement therapies in non-regenerative organs. These therapeutic approaches, however, will critically depend on the purity of the in vitro differentiated cell populations. In particular, remaining undifferentiated hPSC in a transplant can induce teratoma formation. In order to address this challenge, we have developed a laser-based method for the ablation of hPSC from differentiating cell cultures. Specific antibodies were directed against the hPSC surface markers tumor related antigen (Tra)-1-60 and Tra-1-81. These antibodies, in turn, were targeted with nanogold particles. Subsequent laser exposure resulted in a 98,9?±?0,9% elimination of hPSCs within undifferentiated cell cultures. In order to study potential side effects of laser ablation on cells negative for Tra-1-60 and Tra-1-81, hPSC were mixed with GFP-positive hPSC-derived neural precursors (hESCNP) prior to ablation. These studies showed efficient elimination of hPSC while co-treated hESCNP maintained their normal proliferation and differentiation potential. In vivo transplantation of treated and untreated mixed hPSC/hESCNP cultures revealed that laser ablation can dramatically reduce the risk of teratoma formation. Laser-assisted photothermolysis thus represents a novel contact-free method for the efficient elimination of hPSC from in vitro differentiated hPSC-derived somatic cell populations.     

Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells

Precise, robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell?400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension, this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover, an intermediate population of neural precursor cells, which are routinely >95% NCAM^pos and Tra-1-60^neg by FACS analysis, may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments.     

New Approaches in the Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells toward Hepatocytes

Orthotropic liver transplantation is the only established treatment for end-stage liver diseases. Utilization of hepatocyte transplantation and bio-artificial liver devices as alternative therapeutic approaches requires an unlimited source of hepatocytes. Stem cells, especially embryonic stem cells, possessing the ability to produce functional hepatocytes for clinical applications and drug development, may provide the answer to this problem. New discoveries in the mechanisms of liver development and the emergence of induced pluripotent stem cells in 2006 have provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications. This review is aimed towards providing scientists and physicians with the latest advancements in this rapidly progressing field.