Journal of Genetics and Genomics

Stem cell research is coming of age in China

Jianwu Dai, Shaorong Gao

2010, 37(7) doi: 10.1016/S1673-8527(09)60059-X

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Induced pluripotent stem cells (iPSCs)—a new era of reprogramming

Lan Kang, Zhaohui Kou, Yu Zhang, Shaorong Gao

2010, 37(7): 415-421. doi: 10.1016/S1673-8527(09)60060-6

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Embryonic stem cells (ESCs) derived from the early embryos possess two important characteristics: self-renewal and pluripotency, which make ESCs ideal seed cells that could be potentially utilized for curing a number of degenerative and genetic diseases clinically. However, ethical concerns and immune rejection after cell transplantation limited the clinical application of ESCs. Fortunately, the recent advances in induced pluripotent stem cell (iPSC) research have clearly shown that differentiated somatic cells from various species could be reprogrammed into pluripotent state by ectopically expressing a combination of several transcription factors, which are highly enriched in ESCs. This ground-breaking achievement could circumvent most of the limitations that ESCs faced. However, it remains challenging if the iPS cell lines, especially the human iPSCs lines, available are fully pluripotent. Therefore, it is prerequisite to establish a molecular standard to distinguish the better quality iPSCs from the inferior ones.

Contribution of myeloid-derived suppressor cells to tumor-induced immune suppression, angiogenesis, invasion and metastasis

Xian-Zong Ye, Shi-Cang Yu, Xiu-Wu Bian

2010, 37(7): 423-430. doi: 10.1016/S1673-8527(09)60061-8

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Growing evidence suggests that myeloid-derived suppressor cells (MDSCs), which have been named “immature myeloid cells” or “myeloid suppressor cells” (MSCs), play a critical role during the progression of cancer in tumor-bearing mice and cancer patients. As their name implies, these cells are derived from bone marrow and have a tremendous potential to suppress immune responses. Recent studies indicated that these cells also have a crucial role in tumor progression. MDSCs can directly incorporate into tumor endothelium. They secret many pro-angiogenic factors as well. In addition, they play an essential role in cancer invasion and metastasis through inducing the production of matrix metalloproteinases (MMPs), chemoattractants and creating a pre-metastatic environment. Increasing evidence supports the idea that cancer stem cells (CSCs) are responsible for tumorigenesis, resistance to therapies, invasion and metastasis. Here, we hypothesize that CSCs may “hijack” MDSCs for use as alternative niche cells, leading to the maintenance of stemness and enhanced chemo- and radio-therapy resistance. The countermeasure that directly targets to MDSCs may be useful for against angiogenesis and preventing cancer from invasion and metastasis. Therefore, the study of MDSCs is important to understand tumor progression and to enhance the therapeutic efficacy against cancer.

Differentiation of embryonic stem cells in adult bone marrow

Yueying Li, Jing He, Fengchao Wang, Zhenyu Ju, Sheng Liu, Yu Zhang, Zhaohui Kou, Yanfeng Liu, Tao Cheng, Shaorong Gao

2010, 37(7): 431-439. doi: 10.1016/S1673-8527(09)60062-X

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Embryonic stem cells (ESCs) are a potential source of generating transplantable hematopoietic stem and progenitor cells, which in turn can serve as “seed” cells for hematopoietic regeneration. In this study, we aimed to gauge the ability of mouse ESCs directly differentiating into hematopoietic cells in adult bone marrow (BM). To this end, we first derived a new mouse ESC line that constitutively expressed the green fluorescent protein (GFP) and then injected the ESCs into syngeneic BM via intra-tibia. The progeny of the transplanted ESCs were then analyzed at different time points after transplantation. Notably, however, most injected ESCs differentiated into non-hematopoietic cells in the BM whereas only a minority of the cells acquired hematopoietic cell surface markers. This study provides a strategy for evaluating the differentiation potential of ESCs in the BM micro-environment, thereby having important implications for the physiological maintenance and potential therapeutic applications of ESCs.

Role of Lef1 in sustaining self-renewal in mouse embryonic stem cells

Chen Huang, Dajiang Qin

2010, 37(7): 441-449. doi: 10.1016/S1673-8527(09)60063-1

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Embryonic stem cells (ESCs) can self-renew indefinitely while maintaining the ability to generate all three germ-layer derivatives. Despite the importance of ESCs in developmental biology and their potential impact on regenerative medicine, the molecular mechanisms controlling ESC behavior are incompletely understood. Previously, activation of the canonical Wnt signaling pathway has been shown to contribute to mouse ESC self-renewal. Here we report that ectopic expression of Lef1, a component of the Wnt signaling pathway, has a positive effect on the self-renewal of mouse ESCs. Lef1 up-regulates Oct4 promoter activity and physically interacts with Nanog, two key components of the ESC pluripotency machinery. Moreover, siRNA for Lef1 induced mouse ESC differentiation. Our results thus suggest that in response to Wnt signaling Lef1 binds to stabilized β-catenin and helps maintain the undifferentiated status of ESCs through modulation of Oct4 and Nanog.

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds

Jin Zhou, Ye Zhang, Qiuxia Lin, Zhiqiang Liu, Haibin Wang, Cuimi Duan, Yanmeng Wang, Tong Hao, Kuiwu Wu, Changyong Wang

2010, 37(7): 451-460. doi: 10.1016/S1673-8527(09)60064-3

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Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture, they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.

The novel OCT4 spliced variant OCT4B1 can generate three protein isoforms by alternative splicing into OCT4B

Yuan Gao, Xia Wang, Jin Han, Zhifeng Xiao, Bing Chen, Guannan Su, Jianwu Dai

2010, 37(7): 461-465. doi: 10.1016/S1673-8527(09)60065-5

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OCT4 is one of the key transcription factors in maintaining the pluripotency and self-renewal of embryonic stem (ES) cells. HumanOCT4 can generate two isoforms OCT4A and OCT4B by alternative splicing. OCT4B1 is a recently discovered novel OCT4 spliced variant, which has been considered as a putative marker of stemness. Compared with the OCT4B mRNA, OCT4B1 mRNA is generated by retaining intron 2 as a cryptic exon which contains a TGA stop codon in it. As a result, the protein product of OCT4B1 mRNA could be truncated. Interestingly, we present here that OCT4B1 can indirectly produce the same protein products as OCT4B. We have demonstrated that OCT4B1 mRNA can be spliced into OCT4B mRNA, and encode three protein isoforms. The splicing of OCT4B1 mRNA into OCT4B mRNA can be remarkably inhibited by the mutation of the classical splicing site. Our result suggests that OCT4B mRNA may originate from OCT4B1 mRNA by alternative splicing.

Derivation of embryonic stem cells from Brown Norway rats blastocysts

Xiaoyang Zhao, Zhuo Lv, Lei Liu, Liu Wang, Man Tong, Qi Zhou

2010, 37(7): 467-473. doi: 10.1016/S1673-8527(09)60066-7

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Knockout Brown Norway (BN) rat could be a useful disease model for human disorders, however, a failure to derive embryonic stem (ES) cells disturbs the further development of the model. In this study, we reported a case of successful derivation of the BN rat ES cells with the derivation efficiency comparable to that of Sprague Dawley (SD) rats. The BN rat ES cells expressed the key transcription factors, and were able to form embryonic bodies (EBs) when being differentiated in vitro. After injecting the BN rat ES cells into the SD rat blastocysts, high-contribution chimeric rats were generated and could survive to their adulthood. Our success in generating pluripotent rat ES cells will benefit the generation of the knockout rats in the future.

Characterization of OP9 as authentic mesenchymal stem cell line

Jiao Gao, Xin-Long Yan, Ren Li, Yi Liu, Wenyan He, Shengkun Sun, Yu Zhang, Bing Liu, Jiaxiang Xiong, Ning Mao

2010, 37(7): 475-482. doi: 10.1016/S1673-8527(09)60067-9

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Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into various cell types, including osteocytes, chondrocytes, adipocytes, myocytes, and tenocytes. However, the difficulty or failure in expanding the mouse MSCs in vitro greatly hampered important research in animal models. The OP9, a stromal cell line from mouse bone marrow, has hematopoietic supportive capacity. Here, we report that the OP9 has the immunophenotype (CD45⁻, CD11b⁻, FLK-1⁻, CD31⁻, CD34⁻, CD44⁺, CD29⁺, Sca-1⁺, CD86⁻, and MHCII⁻) identical to canonical mouse MSCs. The expression of CD140a⁺, CD140b⁺, α-SMA⁺ and Calponin⁺ suggested the perivascular origin of OP9. Functionally, the OP9 had strong clonogenic ability and could be induced into osteocytes, chondrocytes and adipocytes. The lymphocyte transformation test (LTT) and mixed leukocyte reaction (MLR) showed that the OP9 could suppress T lymphocyte proliferation stimulated by nonspecific mitogens (PHA) or allogeneic lymphocytes (BALB/c T cells). Finally, the migration of OP9 could be efficiently induced by bFGF, IGF-1, IL-3, PDGF-BB, TGF-β1 and TGF-β3. In conclusion, the OP9 were bona fide MSCs, and such homogenous cell line will be helpful to delineate biological features of MSCs at the stem cell level.

Generation and characterisation of rabbit monoclonal antibodies against the native cell surface antigens of embryonic stem cells

Yongliang Lu, Zhongyuan Su, Ying Li, Jingfeng Luo, Zhou Tan, Huijiao Ji, Weimin Zhu, Guoliang Yu, Liangbiao Chen, Ming Zhang

2010, 37(7): 483-492. doi: 10.1016/S1673-8527(09)60068-0

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Embryonic stem (ES) cells are potent resources for cell therapy, and monoclonal antibodies (mAbs) against native cell surface markers of ES cells could be useful tools for therapeutic applications. Here, we report the development of a feasible approach, which could be used in mass production, for experimentally producing rabbit mAbs against native cell surface antigens on the cell surface. Two of the 14 mAbs, which were selected at random, could be bound to the cell surface antigens of mES cells. The immunocytochemistry (ICC) and Western blot results showed that mAb 39 recognises conformational epitopes. The target antigen of mAb 39 was then successfully purified using an improved immunoprecipitation approach in which mAb was bounded to intact mES cells before the cells were lysed. The LC-LTQ mass spectrum analysis showed that the target antigen of mAb 39 was Glut3. This result was further confirmed by Western blot using commercially available antibodies against Glut3. Further experiments showed that mAb 39 exhibited an antiproliferative effect on mES cells. We also found that Glut3 was differentially expressed among the mES cell population as detected by flow cytometry.

2010 Vol. 37, No. 7