The Path from Skin to Brain: Generation of Functional Neurons from Fibroblasts

Cell fate reprogramming makes possible the generation of new cell types from healthy adult cells to replace those lost or damaged in disease. Additionally, reprogramming patient cells into specific cell types allows for drug screening and the development of new therapeutic tools. Generation of new neurons is of particular interest because of the potential to treat diseases of the nervous system, such as neurodegenerative disorders and spinal cord injuries, with cell replacement therapy. Recent advances in cell fate reprogramming have led to the development of novel methods for the direct conversion of fibroblasts into neurons and neural stem cells. This review will highlight the advantages of these new methods over neuronal induction from embryonic stem cells and induced pluripotent stem cells, as well as outline the limitations and the potential for future applications.     

Transitions between epithelial and mesenchymal states during cell fate conversions

Cell fate conversion is considered as the changing of one type of cells to another type including somatic cell reprogramming (de-differentiation), differentiation, and trans-differentiation. Epithelial and mesenchymal cells are two major types of cells and the transitions between these two cell states as epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) have been observed during multiple cell fate conversions including embryonic development, tumor progression and somatic cell reprogramming. In addition, MET and sequential EMT-MET during the generation of induced pluripotent stem cells (iPSC) from fibroblasts have been reported recently. Such observation is consistent with multiple rounds of sequential EMT-MET during embryonic development which could be considered as a reversed process of reprogramming at least partially. Therefore in current review, we briefly discussed the potential roles played by EMT, MET, or even sequential EMT-MET during different kinds of cell fate conversions. We also provided some preliminary hypotheses on the mechanisms that connect cell state transitions and cell fate conversions based on results collected from cell cycle, epigenetic regulation, and stemness acquisition.     

Pluripotent stem cells induced from mouse neural stem cells and small intestinal epithelial cells by small molecule compounds

Recently, we reported a chemical approach to generate pluripotent stem cells from mouse fibroblasts. However, whether chemically induced pluripotent stem cells (CiPSCs) can be derived from other cell types remains to be demonstrated. Here, using lineage tracing, we first verify the generation of CiPSCs from fibroblasts. Next, we demonstrate that neural stem cells (NSCs) from the ectoderm and small intestinal epithelial cells (IECs) from the endoderm can be chemically reprogrammed into pluripotent stem cells. CiPSCs derived from NSCs and IECs resemble mouse embryonic stem cells in proliferation rate, global gene expression profile, epigenetic status, self-renewal and differentiation capacity, and germline transmission competency. Interestingly, the pluripotency gene Sall4 is expressed at the initial stage in the chemical reprogramming process from different cell types, and the same core small molecules are required for the reprogramming, suggesting conservation in the molecular mechanism underlying chemical reprogramming from these diverse cell types. Our analysis also shows that the use of these small molecules should be fine-tuned to meet the requirement of reprogramming from different cell types. Together, these findings demonstrate that full chemical reprogramming approach can be applied in cells of different tissue origins and suggest that chemical reprogramming is a promising strategy with the potential to be extended to more initial types.     

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.     

Cellular reprogramming for clinical cartilage repair

The repair of articular cartilage needs a sufficient number of chondrocytes to replace the defect tissue, and therefore, expansion of cells is generally required. Chondrocytes derived by cellular reprogramming may provide a solution to the limitations of current (stem) cell-based therapies. In this article, two distinct approaches—induced pluripotent stem cell (iPSC)-mediated reprogramming and direct lineage conversion—are analysed and compared according to criteria that encompass the qualification of the method and the derived chondrocytes for the purpose of clinical application. Progress in iPSC generation has provided insights into the replacement of reprogramming factors by small molecules and chemical compounds. As follows, multistage chondrogenic differentiation methods have shown to improve the chondrocyte yield and quality. Nevertheless, the iPSC ‘detour’ remains a time- and cost-consuming approach. Direct conversion of fibroblasts into chondrocytes provides a slight advantage over these aspects compared to the iPSC detour. However, the requirement of constitutive transgene expression to inhibit hypertrophic differentiation limits this approach of being translated to the clinic. It can be concluded that the quality of the derived chondrocytes highly depends on the characteristics of the reprogramming method and that this is important to keep in mind during the experimental set-up. Further research into both reprogramming approaches for clinical cartilage repair has to include proper control groups and epigenetic profiling to optimize the techniques and eventually derive functionally stable articular chondrocytes.     

Non-Genetic Direct Reprogramming and Biomimetic Platforms in a Preliminary Study for Adipose-Derived Stem Cells into Corneal Endothelia-Like Cells

Cell fate and function can be regulated and reprogrammed by intrinsic genetic program, extrinsic factors and niche microenvironment. Direct reprogramming has shown many advantages in the field of cellular reprogramming. Here we tried the possibility to generate corneal endothelia (CE) -like cells from human adipose-derived stem cells (ADSCs) by the non-genetic direct reprogramming of recombinant cell-penetrating proteins Oct4/Klf4/Sox2 (PTD-OKS) and small molecules (purmorphamine, RG108 and other reprogramming chemical reagents), as well as biomimetic platforms of simulate microgravity (SMG) bioreactor. Co-cultured with corneal cells and decellularized corneal ECM, Reprogrammed ADSCs revealed spherical growth and positively expressing Nanog for RT-PCR analysis and CD34 for immunofluorescence staining after 7 days-treatment of both purmorphamine and PTD-OKS (P-OKS) and in SMG culture. ADSCs changed to CEC polygonal morphology from spindle shape after the sequential non-genetic direct reprogramming and biomimetic platforms. At the same time, induced cells converted to weakly express CD31, AQP-1 and ZO-1. These findings demonstrated that the treatments were able to promote the stem-cell reprogramming for human ADSCs. Our study also indicates for the first time that SMG rotary cell culture system can be used as a non-genetic means to promote direct reprogramming. Our methods of reprogramming provide an alternative strategy for engineering patient-specific multipotent cells for cellular plasticity research and future autologous CEC replacement therapy that avoids complications associated with the use of human pluripotent stem cells.     

Partial reprogramming as a therapeutic approach for heart disease: A state-of-the-art review

Heart disease such as myocardial infarction is the first cause of mortality in all countries. Today, cardiac cell-based therapy using de novo produced cardiac cells is considered as a novel approach for cardiac regenerative medicine. Recently, an alchemy-like approach, known as direct reprogramming or direct conversion, has been developed to directly convert somatic cells to cardiac cells in vitro and in vivo. This cellular alchemy is a short-cut and safe strategy for generating autologous cardiac cells, and it can be accomplished through activating cardiogenesis- or pluripotency-related factors in noncardiac cells. Importantly, pluripotency factors-based direct cardiac conversion, known as partial reprogramming, is shorter and more efficient for cardiomyocyte generation in vitro. Today, this strategy is achievable for direct conversion of mouse and human somatic cells to cardiac lineage cells (cardiomyocytes and cardiac progenitor cells), using transgene free, chemical-based approaches. Although, heart-specific partial reprogramming seems to be challenging for in vivo conversion of cardiac fibroblasts to cardiac cells, but whole organism-based in vivo partial reprogramming ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in mouse. Notably, cardiac cells produced using partial reprogramming strategy can be a useful platform for disease modeling, drug screening and cardiac cell-based therapy, once the safety issues are overcome. Herein, we discuss about all progresses in de novo production of cardiac cells using partial reprogramming-based direct conversion, as well as give an overview about the potential applications of this strategy in vivo and in vitro.     

Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression. These small, non-coding RNAs are believed to regulate more than a third of all protein coding genes, and they have been implicated in the control of virtually all biological processes, including the biology of stem cells. The essential roles of miRNAs in the control of pluripotent stem cells were clearly established by the finding that embryonic stem (ES) cells lacking proteins required for miRNA biogenesis exhibit defects in proliferation and differentiation. Subsequently, the function of numerous miRNAs has been shown to control the fate of ES cells and to directly influence critical gene regulatory networks controlled by pluripotency factors Sox2, Oct4, and Nanog. Moreover, a growing list of tissue-specific miRNAs, which are silenced or not processed fully in ES cells, has been found to promote differentiation upon their expression and proper processing. The importance of miRNAs for ES cells is further indicated by the exciting discovery that specific miRNA mimics or miRNA inhibitors promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Although some progress has been made during the past two years in our understanding of the contribution of specific miRNAs during reprogramming, further progress is needed since it is highly likely that miRNAs play even wider roles in the generation of iPS cells than currently appreciated. This review examines recent developments related to the roles of miRNAs in the biology of pluripotent stem cells. In addition, we posit that more than a dozen additional miRNAs are excellent candidates for influencing the generation of iPS cells as well as for providing new insights into the process of reprogramming.     

体细胞重编程为多能干细胞的研究进展

体细胞通过重编程转变成其他类型的细胞,在再生医学方面具有重要的应用前景。细胞重编程的方法主要有体细胞核移植、细胞融合、细胞提取物诱导、限定因子诱导等,这些方法可以不同程度地改变细胞命运。最近,限定因子诱导的多能干细胞（induced pluripotent stem cell。iPS）为重编程提供了一种崭新的方法,不仅可以避免伦理争议,还提供了一种更为便利的技术,为再生医学开辟了新的天地;同时,iPS技术为研究基因表达调控、蛋白质互作、机体生长发育等提供了一个非常重要的研究手段。本文主要论述了体细胞重编程的方法及iPS细胞的进展、面临的问题和应用前景。     

Direct lineage conversions: unnatural but useful?

Classic experiments such as somatic cell nuclear transfer into oocytes and cell fusion demonstrated that differentiated cells are not irreversibly committed to their fate. More recent work has built on these conclusions and discovered defined factors that directly induce one specific cell type from another, which may be as distantly related as cells from different germ layers. This suggests the possibility that any specific cell type may be directly converted into any other if the appropriate reprogramming factors are known. Direct lineage conversion could provide important new sources of human cells for modeling disease processes or for cellular-replacement therapies. For future applications, it will be critical to carefully determine the fidelity of reprogramming and to develop methods for robustly and efficiently generating human cell types of interest.