细胞重编程中的表观遗传分子机制

成体细胞可以通过核移植、细胞融合或者特定因子导入的方式实现重编程回到多能性状态。在重编程的过程中,表观遗传水平的调控机制起到了非常关键的作用。通过回顾重编程的研究进展来探讨表观遗传学在重编程中的调控机制。     

中国细胞重编程和多能干细胞研究进展

近几十年是干细胞领域飞速发展的重要时期。随着中国经济实力的发展壮大,科研实力也在稳步增强,干细胞研究领域达到了国际并跑甚至领跑的水平。本文从体细胞核移植、诱导多能干细胞、单倍体多能干细胞和胚胎早期发育研究4个方面,对中国细胞重编程和干细胞领域的研究进展进行了历史性回顾,总结了中国科学家在相关领域所取得的重要科研成果。随着单细胞测序技术的发展,各种发育过程将实现更为深入的解读,干细胞的临床应用在中国也会大放异彩。     

细胞重编程的分子机制

细胞重编程,尤其是诱导多能性干细胞的出现,给再生医学带来极大的希望。近年来,这方面的研究吸引了众多科学家的参与,也取得了非常丰富的成果。本文主要从转录因子、表观遗传和信号转导等角度,介绍了细胞重编程分子机制研究方面的进展和未来的方向。     

Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming

Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell‐like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real‐time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration‐free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.     

Genetic background affects susceptibility to tumoral stem cell reprogramming

The latest studies of the interactions between oncogenes and its target cell have shown that certain oncogenes may act as passengers to reprogram tissue-specific stem/progenitor cell into a malignant cancer stem cell state. In this study, we show that the genetic background influences this tumoral stem cell reprogramming capacity of the oncogenes using as a model the Sca1-BCRABLp210 mice, where the type of tumor they develop, chronic myeloid leukemia (CML), is a function of tumoral stem cell reprogramming. Sca1-BCRABLp210 mice containing FVB genetic components were significantly more resistant to CML. However, pure Sca1-BCRABLp210 FVB mice developed thymomas that were not seen in the Sca1-BCRABLp210 mice into the B6 background. Collectively, our results demonstrate for the first time that tumoral stem cell reprogramming fate is subject to polymorphic genetic control.     

The neurosteroid dehydroepiandrosterone could improve somatic cell reprogramming

Expression of four major reprogramming transgenes, including Oct4, Sox2, Klf4 and c-myc, in somatic cells enables them to have pluripotency. These cells are iPSC (induced pluripotent stem cell) that currently show the greatest potential for differentiation into cells of the three germ lineages. One of the issues facing the successful reprogramming and clinical translation of iPSC technology is the high rate of apoptosis after the reprogramming process. Reprogramming is a stressful process, and the p53 apoptotic pathway plays a negative role in cell growth and self-renewal. Apoptosis via the p53 pathway serves as a major barrier in nuclear somatic cell reprogramming during iPSC generation. DHEA (dehydroepiandrosterone) is an abundant steroid that is produced at high levels in the adrenal cells, and withdrawal of DHEA increases the levels of p53 in the epithelial and stromal cells, resulting in increased levels of apoptotic cells; meanwhile, DHEA decreases cellular apoptosis. DHEA could improve the efficacy of reprogramming yield due to a decrease in apoptosis via the p53 pathway and an increase in cell viability.     

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。     

Three-dimensional cell culture models for investigating human viruses

Three-dimensional (3D) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. Moreover, these models bridge the gap between traditional two-dimensional (2D) monolayer cultures and animal models. 3D culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. In addition, 3D models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. This review summarizes and analyzes recent progress in human virological research with 3D cell culture models. We discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3D culture models.

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The evolving biology of cell reprogramming

Modern stem cell biology has achieved a transformation that was thought by many to be every bit as unattainable as the ancient alchemists' dream of transforming base metals into gold. Exciting opportunities arise from the process known as 'cellular reprogramming' in which cells can be reliably changed from one tissue type to another. This is enabling novel approaches to more deeply investigate the fundamental basis of cell identity. In addition, new opportunities have also been created to study (perhaps even to treat) human genetic and degenerative diseases. Specific cell types that are affected in inherited disease can now be generated from easily accessible cells from the patient and compared with equivalent cells from healthy donors. The differences in cellular phenotype between the two may then be identified, and assays developed to establish therapies that prevent the development or progression of disease symptoms. Cellular reprogramming also has the potential to create new cells to replace those whose death or dysfunction causes disease symptoms. For patients suffering from inherited cases of degenerative diseases like Parkinson's disease or amyotrophic lateral sclerosis (also known as motor neuron disease), the future realization of such cell-based therapies would truly be worth its weight in gold. However, before this enormous potential can become a reality, several significant biological and technical challenges must be overcome. Furthermore, to maintain the credibility of the scientific community with the general public, it is important that hope-inspiring advances are not over-hyped. The papers in this issue of the Philosophical Transactions of the Royal Society B: Biological Sciences cover many areas relevant to this topic. In this Introduction, we provide an overall context in which to consider these individual papers.     

Three-dimensional biomimetic hyaluronic acid hydrogels to investigate glioblastoma stem cell behaviors

Glioblastoma multiforme (GBM) is the deadliest form of primary brain tumor. GBM tumors are highly heterogeneous, being composed of tumor cells as well as glioblastoma stem cells (GSCs) that contribute to drug resistance and tumor recurrence following treatment. To develop therapeutic strategies, an improved understanding of GSC behavior in their microenvironment is critical. Herein, we have employed three-dimensional (3D) hyaluronic acid (HA) hydrogels that allow the incorporation of brain microenvironmental cues to investigate GSC behavior. U87 cell line and patient-derived D456 cells were cultured as suspension cultures (serum-free) and adherently (in the presence of serum) and were then encapsulated in HA hydrogels. We observed that all the seeded single cells expanded and formed spheres, and the size of the spheres increased with time. Increasing the initial cell seeding density of cells influenced the sphere size distribution. Interestingly, clonal expansion of serum-free grown tumor cells in HA hydrogels was observed. Also, stemness marker expression of serum and/or serum-free grown cells was altered when cultured in HA hydrogels. Finally, we demonstrated that HA hydrogels can support long-term GSC culture (up to 60 days) with retention of stemness markers. Overall, such biomimetic culture systems could further our understanding of the microenvironmental regulation of GSC phenotypes.     

Dysfunctional mitochondrial fission impairs cell reprogramming

We have recently shown that mitochondrial fission is induced early in reprogramming in a Drp1-dependent manner; however, the identity of the factors controlling Drp1 recruitment to mitochondria was unexplored. To investigate this, we used a panel of RNAi targeting factors involved in the regulation of mitochondrial dynamics and we observed that MiD51, Gdap1 and, to a lesser extent, Mff were found to play key roles in this process. Cells derived from Gdap1-null mice were used to further explore the role of this factor in cell reprogramming. Microarray data revealed a prominent down-regulation of cell cycle pathways in Gdap1-null cells early in reprogramming and cell cycle profiling uncovered a G2/M growth arrest in Gdap1-null cells undergoing reprogramming. High-Content analysis showed that this growth arrest was DNA damage-independent. We propose that lack of efficient mitochondrial fission impairs cell reprogramming by interfering with cell cycle progression in a DNA damage-independent manner.     

细胞重编程与表观遗传学调控

细胞重编程是生命科学研究的热点之一,目前体细胞核移植、细胞融合和特定转录因子诱导等方法都可以实现体外细胞重编程,而在细胞重编程过程中表观遗传学发挥关键的调控作用,因此对重编程过程中表观遗传学调控机制开展深入研究具有重要的意义。本文简要综述细胞重编程的研究现状和表观遗传学调控细胞重编程机制的研究进展,并对小分子化合物和microRNA提高细胞重编程效率的最新进展进行了介绍。