7月，最新一期Cell Stem Cell杂志发表了3个研究团队的最新研究成果，研究者成功的将人体血细胞转化为iPS细胞。专家称这些研究加快了干细胞技术应用。日本京都大学科学家山中伸弥（Shinya Yamanaka）特别为本期Cell Stem Cell发表评论文章称这三项研究是干细胞领域发展的巨大进步。
三年前，来自日本和美国的研究人员称他们已经将人的表皮细胞转化为iPS细胞。这周，3个研究小组称从血液中获得了iPS细胞。此前人们通常采集皮肤细胞来制作iPS细胞，耗时长达六七十天。新技术极大地缩短了时间，从血液采集算起只需要25天。新方法可以使用验血时采集的血液。研究者将目光瞄准了血液中富含的一种淋巴细胞“T细胞”，从0.1毫升的血液中提取T细胞，再通过“仙台病毒”将制作iPS细胞所需的4种特定基因导入T细胞中。该病毒即使感染了细胞，也只会停留在细胞质中，不会侵入细胞核，因此不会损伤细胞DNA，具有细胞癌变风险低的优点。研究人员已确认，制成的iPS细胞中不含仙台病毒，可以分化成各种体细胞。
美国宾尼法尼亚州大学再生医学研究所John Gearhart博士称，这三篇文章对于iPS领域来说非常重要，血液细胞转化获得iPS细胞，这为研究者和患者提供了便利。
Patient-Specific Pluripotent Stem Cells Become Even More Accessible
Shinya Yamanaka
1 Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
2 Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA
In this issue of Cell Stem Cell, Staerk et al. (2010), Seki et al. (2010),Staerk et al. (2010), Seki et al. (2010), and Loh et al. (2010) each describe the derivation of human iPSCs from peripheral blood. Although seemingly incremental, this advance brings the stem cell field an important step closer to eventual clinical use.
生物谷推荐Tomohisa Seki等发表文章的索引
Cell Stem Cell>doi:10.1016/j.stem.2010.06.003
Generation of Induced Pluripotent Stem Cells from Human Terminally Differentiated Circulating T Cells
Tomohisa Seki1, 7, Shinsuke Yuasa1, 2, 7, Mayumi Oda2, Toru Egashira1, Kojiro Yae1, Dai Kusumoto1, Hikari Nakata1, Shugo Tohyama1, Hisayuki Hashimoto1, Masaki Kodaira1, Yohei Okada2, 3, Hiroyuki Seimiya4, Noemi Fusaki5, 6, Mamoru Hasegawa5 and Keiichi Fukuda1
1 Department of Cardiology, Keio University School of Medicine, Tokyo 160-8582, Japan
2 Center for Integrated Medical Research, Keio University School of Medicine, Tokyo 160-8582, Japan
3 Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
4 Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan
5 DNAVEC Corporation, Ibaraki 1-25-11, Japan
6 PRESTO, JST, Saitama 332-0012, Japan
The direct reprogramming of somatic cells to produce induced pluripotent stem cells (iPSCs) is a prominent recent advance in stem cell biology (Takahashi and Yamanaka, 2006). Generation of iPSCs without genomic integration of extrinsic genes is highly desirable. Initially, human dermal fibroblasts were used to derive human iPSCs (hiPSCs) (Takahashi et al., 2007,Yu et al., 2007). However, recent studies have shown that other human somatic stem cells can be used (Aasen et al., 2008,Eminli et al., 2009,Kim et al., 2009,Ye et al., 2009). It is difficult to obtain human somatic stem cells, but human terminally differentiated circulating T cells (hTDCTCs) are readily available from peripheral blood. Here, we show that a combination of activated T cell cultivation and a temperature-sensitive mutated Sendai virus (SeV) that encodes human OCT3/4, SOX2, KLF4, and c-MYC allows the generation of hiPSCs easily, efficiently, and safely within a 1 month time frame.
Sampling of peripheral blood is one of the least invasive procedures performed routinely in clinics, and surplus peripheral blood samples are often left unused after clinical examinations. Among peripheral blood mononuclear cells (PBMCs), T cells can be readily cultured in vitro by means of a plate-bound anti-CD3 monoclonal antibody and recombinant (r)IL-2 (Desai-Mehta et al., 1996), and we used such an approach to expand hTDCTCs from peripheral blood samples. From 1 ml of whole blood, PBMCs were separated on a Ficoll gradient and then cultured with plate-bound anti-CD3 monoclonal antibody and rIL-2 (Figure 1A). Although PBMC fractions contain lymphocytes and monocytes, T cells are selectively cultured under these conditions. In culture, the number of activated T cells increased gradually but consistently. Five days after blood sampling, the cultured cells were morphologically identical to pure CD3-positive T cells collected by fluorescence-activated cell sorting (FACS) (Figure 1B). We used a whole-PBMC culture method because it is technically simpler than FACS, in which the sorted cells are frequently damaged by laser emission and the process of single-cell sorting.
生物谷推荐Yuin-Han Loh等发表文章的索引
Cell Stem Cell doi:10.1016/j.stem.2010.06.004
Reprogramming of T Cells from Human Peripheral Blood
Yuin-Han Loh1, 2, Odelya Hartung1, 2, Hu Li3, 4, Chunguang Guo5, 6, 7, Julie M. Sahalie1, 2, Philip D. Manos1, 2, Achia Urbach1, 2, Garrett C. Heffner1, 2, Marica Grskovic8, Francois Vigneault7, M. William Lensch1, 2, 5, In-Hyun Park1, 2, Suneet Agarwal1, 2, George M. Church7, James J. Collins3, 4, 5, Stefan Irion8, , and George Q. Daley1, 2, 5, 9, 10
Stem Cell Transplantation Program, Division of Pediatric Hematology Oncology, Children's Hospital Boston and Dana-Farber Cancer Institute; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
2 Harvard Stem Cell Institute, Cambridge, MA 02138, USA
3 Department of Biomedical Engineering and Center for BioDynamics, Boston University, Boston, MA 02215, USA
4 Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
5 Howard Hughes Medical Institute, Boston, MA 02115, USA
6 Immune Diseases Institute, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA
7 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
8 iPierian, Inc. South San Francisco, CA 94080, USA
9 Division of Hematology, Brigham and Women's Hospital, Boston, MA 02115, USA
10 Manton Center for Orphan Disease Research, Boston, MA 02115, USA
Human induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for modeling human diseases. Dermal fibroblasts are frequently used for reprogramming, but require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Here, we report the derivation of iPSCs from multiple human blood sources including peripheral blood mononuclear cells (PBMCs) harvested by routine venipuncture. Peripheral blood-derived human iPSC lines are comparable to human embryonic stem cells (ESCs) with respect to morphology, expression of surface antigens, activation of endogenous pluripotency genes, DNA methylation, and differentiation potential. Analysis of immunoglobulin and T cell receptor gene rearrangement revealed that some of the PBMC iPSCs were derived from T cells, documenting derivation of iPSCs from terminally differentiated cell types. Importantly, peripheral blood cells can be isolated with minimal risk to the donor and can be obtained in sufficient numbers to enable reprogramming without the need for prolonged expansion in culture. Reprogramming from blood cells thus represents a fast, safe, and efficient way of generating patient-specific iPSCs.
Somatic cells can be induced to the pluripotent state by the enforced expression of several transcription factors including OCT4, SOX2, KLF4, MYC, NANOG, and LIN28 (Takahashi et al., 2007,Yu et al., 2007,Park et al., 2008a). Human iPSCs are commonly generated from dermal fibroblasts harvested by surgical skin biopsy (Park et al., 2008b). Exposure of the dermis to ultraviolet light increases the risk for chromosomal aberrations (Ikehata et al., 2003), raising concerns for whether iPSCs will reflect the patient's constitutional genotype. For routine clinical application, it would be desirable to reprogram cell types that are safe and can be collected noninvasively in large numbers.
Blood is a cell source that can be easily obtained from patients. Mouse B and T cells are amenable to reprogramming by overexpressing Oct4, Sox2, Klf4, and Myc with the ectopic expression of Cepbα andp53>knockdown, respectively (Hanna et al., 2008,Hong et al., 2009). iPSC lines have also been generated from mouse bone marrow progenitor cells (Okabe et al., 2009). We have previously reprogrammed cytokine-mobilized human CD34+ peripheral blood cells to pluripotency, but such harvests are cumbersome, expensive, and time consuming (Loh et al., 2009). Several recent studies reported the generation of iPSCs from human bone marrow and cord blood (Ye et al., 2009,Giorgetti et al., 2009,Haase et al., 2009), but bone marrow harvesting is an invasive procedure, and cord blood is available for only a minority of individuals who have their samples banked at birth. A recent study with peripheral blood from donors with myeloproliferative disorder (MPD) isolated iPSC colonies that contain the JAK2-V617F mutation (Ye et al., 2009), but MPD is characterized by abnormally high numbers of circulating CD34+ cells from the bone marrow. These previous studies demonstrating successful reprogramming of blood cells into iPSCs have relied on specialized blood cell sources with high proliferative potential.
CD34+ hematopoietic stem/progenitor cells mobilized into the donor's peripheral blood by pretreatment with granulocyte colony-stimulating factor (G-CSF) can be successfully reprogrammed to pluripotency (Loh et al., 2009). To test whether we can reprogram cells from routine peripheral blood (PB) sources, we obtained CD34+ purified blood samples from a healthy 49-year-old male donor who had undergone simple apheresis without cytokine priming. We also isolated mononuclear cells (PBMCs) from the peripheral blood samples collected by venipuncture of four healthy donors (28- to 49-years-old) via Ficoll density centrifugation.
To induce reprogramming of enriched CD34+ blood cells, we infected with lentiviruses expressing OCT4, SOX2, KLF4, and MYC reprogramming factors (Figure 1A). Colonies with well-defined hESC-like morphology were first observed 21 days after transduction (Figure 1B). For reprogramming of fresh peripheral blood mononuclear cells (PBMCs), we employed two rounds of lentiviral infection (day 0 and day 8) and isolated colonies with distinct flat and compact morphology with clear-cut round edges reminiscent of hESCs after a slightly longer latency of around 35 days (Figure 1C). Interestingly, a previous study with a single round of lentiviral infection of PBMCs failed to observe iPSC colony formation (Haase et al., 2009). In a separate set of experiments, we tested the ability of retroviruses encoding the human reprogramming factors to generate iPSCs from human PBMCs, and despite low infection efficiency, we observed iPSC colonies after 25–35 days (Figure 1D).
生物谷推荐Judith Staerk等文章的索引：
Cell Stem Cell>doi:10.1016/j.stem.2010.06.002
Reprogramming of Human Peripheral Blood Cells to Induced Pluripotent Stem Cells
Judith Staerk1, Meelad M. Dawlaty1, Qing Gao1, Dorothea Maetzel1, Jacob Hanna1, Cesar A. Sommer2, Gustavo Mostoslavsky2 and Rudolf Jaenisch1, 3, ,>
1 The Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
2 Section of Gastroenterology, Department of Medicine and Center for Regenerative Medicine (CReM), Boston University School of Medicine, Boston, MA 02118, USA
3 Department of Biology, Massachusetts Institute of Technology, 31 Ames Street, Cambridge, MA 02139, USA
Embryonic stem cells are pluripotent cells derived from the inner cell mass of the developing embryo that have the capacity to differentiate into every cell type of the adult (Evans and Kaufman, 1981,Martin, 1981,Martin and Evans, 1975,Thomson et al., 1998). The generation of patient-specific pluripotent cells is therefore an important goal of regenerative medicine. A major step to achieve this was the recent discovery that ectopic expression of defined transcription factors induces pluripotency in somatic cells (Lowry et al., 2008,Park et al., 2008b,Takahashi et al., 2007,Yu et al., 2007). Until now, the most common source from which to derive human iPSCs has been skin fibroblasts (Lowry et al., 2008,Park et al., 2008a,Park et al., 2008b,Takahashi et al., 2007,Yu et al., 2009). However, the requirement for skin biopsies and the need to expand fibroblast cells for several passages in vitro represent a hurdle that must be overcome to make iPSC technology broadly applicable. Peripheral blood can be utilized as an easily accessible source of patient tissue for reprogramming. Here we derived iPSCs from frozen human peripheral blood samples. Some of the iPSCs had rearrangements of the T cell receptor (TCR), indicating that T cells can be reprogrammed to pluripotency.
Recently, granulocyte colony stimulating factor (G-CSF)-mobilized CD34+ blood cells have been used as a source from which to derive iPSCs (Loh et al., 2009). However, this requires the subcutaneous injection of G-CSF, a process that can be applied only if the donor is in good medical condition. Also, the negative effects of treatment of patients with growth factors such as erythropoietin (Miller et al., 2009) and G-CSF are still being investigated. Of concern is the use of G-CSF because this cytokine is a growth factor for myeloid cell precursors (Touw and van de Geijn, 2007) and because G-CSF treatment of patients with severe congenital neutropenia (SCN) can result in a truncated G-CSF receptor allele and acute myeloid leukemia transformation (Touw, 1997). Derivation of iPSCs from peripheral mononuclear blood cells would circumvent all these issues; in addition, peripheral blood is the most accessible adult tissue and permits access to numerous frozen samples already stored at blood banks. Such samples could be expanded in culture and reprogrammed to iPSCs, which in turn allows researchers to study the molecular mechanism underlying blood and other disorders. We show here the derivation of iPSC clones from mature peripheral blood T and myeloid cells.
Mononuclear (MNC) blood cells were isolated from several donors by Ficoll-Hypaque density gradient centrifugation (Ferrante and Thong, 1980,Vissers et al., 1988). Samples were frozen and thawed days to several months after freezing and expanded in IL-7 or in G-CSF, GM-CSF, IL-6, and IL-3 for 5 days. In our initial experiments we used the FUW-M2rtTA and the individual doxycycline-inducible lentiviruses encoding Oct4, Sox2, c-Myc, or Klf4 (Brambrink et al., 2008). However, in ten independent experiments we were not able to reprogram peripheral blood cells with this system. One possibility for failure to obtain iPSCs is that peripheral blood cells are difficult to infect, reducing the probability of obtaining cells carrying the four factors as well as the FUW-M2rtTA construct. Also, we find that the efficiency of blood reprogramming (0.001%–0.0002%) is approximately 10–50 times lower than that of human fibroblast reprogramming.
To increase the infection efficiency, we used a doxycycline-inducible lentivirus encoding all four factors Oct4, Klf4, Sox2, and c-Myc from a polycistronic expression cassette (pHAGE2-TetOminiCMV-hSTEMCCA) (Sommer et al., 2010). Blood cells were simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator (FUW-M2rtTA) (Hockemeyer et al., 2008) as well as the polycistronic vector. Infected blood cells were transferred onto feeder layers of mouse embryonic fibroblasts (MEFs) and cultured in the presence of IL-7 or G-CSF, GM-CSF, IL-6, and IL-3 and 2 μg/ml doxycycline (Dox) for an additional 4 days (Figure 1A). At day 5 after Dox induction, the cells were transferred to human ESC medium containing 2 μg/ml Dox, and 25–40 days later colonies were picked and expanded.
