我在考研网上也发了本文，望更多朋友注意！
自从2007年日本的科学家发明了iPS技术以来，关于iPS的研究可谓炙手可热。从2008年始，每年的考研试题，特别是各大名校和研究所，都一定会考到相交内容。在这里我作一专题介绍，引用一些来自网上的资料(还可以附上几个论文)。权当抛砖引玉。
2010年考研的朋友，一定提前看看噢！
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技术路线
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如下图所示(对应的论文摘要如下):
Nature , | doi:10.1038/nature07404; Received 1 February 2008; Accepted 18 September 2008; Published online 8 October 2008
Generation of pluripotent stem cells from adult human testis
Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and show similar properties to embryonic stem cells. Here we report the successful establishment of human adult germline stem cells derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of these cells revealed many similarities to human embryonic stem cells, and the germline stem cells produced teratomas after transplantation into immunodeficient mice. The human adult germline stem cells differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of human embryonic stem cells. We conclude that the generation of human adult germline stem cells from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.
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一般来说，iPS需要四种基因，Klf4/6, Oct3/4, Sox1/2和c-myc。(这个题目今年中科院细胞的第一个大题考到，分值15分！)其中c-myc 是原癌基因。关于这些基因的简要介绍，请参照下文Genes of induction部分。
另外，要确定这些基因用病毒载体或腺病毒载体等方面导入上皮细胞等成体后，是否表现出了干细胞的特点，有一系列的方法。具体请参照下文Identity部分
Genes of induction
The generation of iPS cells is crucial on the genes used for the induction.
Oct-3/4 and certain members of the Sox gene family (Sox1, Sox2, Sox3, and Sox15) have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible. Additional genes, however, including certain members of the Klf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (C-myc, L-myc, and N-myc), Nanog, and LIN28, have been identified to increase the induction efficiency.
* Oct-3/4 (Pou5f1): Oct-3/4 is one of the family of octamer ("Oct") transcription factors, and plays a crucial role in maintaining pluripotency. The absence of Oct-3/4 in Oct-3/4+ cells, such as blastomeres and embryonic stem cells, leads to spontaneous trophoblast differentiation, and presence of Oct-3/4 thus gives rise to the pluripotency and differentiation potential of embryonic stem cells. Various other genes in the "Oct" family, including Oct-3/4's close relatives, Oct1 and Oct6, fail to elicit induction, thus demonstrating the exclusiveness of Oct-3/4 to the induction process.
* Sox family: The Sox family of genes is associated with maintaining pluripotency similar to Oct-3/4, although it is associated with multipotent and unipotent stem cells in contrast with Oct-3/4, which is exclusively expressed in pluripotent stem cells. While Sox2 was the initial gene used for induction by Yamanaka et al., Jaenisch et al., and Thompson et al., other genes in the Sox family have been found to work as well in the induction process. Sox1 yields iPS cells with a similar efficiency as Sox2, and genes Sox3, Sox15, and Sox18 also generate iPS cells, although with decreased efficiency.
* Klf family: Klf4 of the Klf family of genes was initially identified by Yamanaka et al. and confirmed by Jaenisch et al. as a factor for the generation of mouse iPS cells and was demonstrated by Yamanaka et al. as a factor for generation of human iPS cells. However, Thompson et al. reported that Klf4 was unnecessary for generation of human iPS cells and in fact failed to generate human iPS cells. Klf2 and Klf4 were found to be factors capable of generating iPS cells, and related genes Klf1 and Klf5 did as well, although with reduced efficiency.
* Myc family: The Myc family of genes are proto-oncogenes implicated in cancer. Yamanaka et al. and Jaenisch et al. demonstrated that c-myc is a factor implicated in the generation of mouse iPS cells and Yamanaka et al. demonstrated it was a factor implicated in the generation of human iPS cells. However, Thomson et al., Yamanaka et al., and unpublished work by Johns Hopkins University reported that c-myc was unnecessary for generation of human iPS cells. Usage of the "myc" family of genes in induction of iPS cells is troubling for the eventuality of iPS cells as clinical therapies, as 25% of mice transplanted with c-myc-induced iPS cells developed lethal teratomas. N-myc and L-myc have been identified to induce instead of c-myc with similar efficiency.
* Nanog: In embryonic stem cells, Nanog, along with Oct-3/4 and Sox2, is necessary in promoting pluripotency. Therefore, it was surprising when Yamanaka et al. reported that Nanog was unnecessary for induction although Thomson et al. has reported it is possible to generate iPS cells with Nanog as one of the factors.
* LIN28: LIN28 is an mRNA binding protein expressed in embryonic stem cells and embryonic carcinoma cells associated with differentiation and proliferation. Thomson et al. demonstrated it is a factor in iPS generation, although it is unnecessary.
Identity
The generated iPSCs were remarkably similar to naturally-isolated pluripotent stem cells (such as mouse and human embryonic stem cells, mESCs and hESCs, respectively) in the following respects, thus confirming the identity, authenticity, and pluripotency of iPSCs to naturally-isolated pluripotent stem cells:
* Cellular biological properties
o Morphology: iPSCs were morphologically similar to ESCs. Each cell had round shape, large nucleolus and scant cytoplasm. Colonies of iPSCs were also similar to that of ESCs. Human iPSCs formed sharp-edged, flat, tightly-packed colonies similar to hESCs and mouse iPSCs formed the colonies similar to mESCs, less flatter and more aggregated colonies than that of hESCs.
o Growth properties: Doubling time and mitotic activity are cornerstones of ESCs, as stem cells must self-renew as part of their definition. iPSCs were mitotically active, actively self-renewing, proliferating, and dividing at a rate equal to ESCs.
o Stem Cell Markers: iPSCs expressed cell surface antigenic markers expressed on ESCs. Human iPSCs expressed the markers specific to hESC, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog. Mouse iPSCs expressed SSEA-1 but not SSEA-3 nor SSEA-4, similarly to mESCs.
o Stem Cell Genes: iPSCs expressed genes expressed in undifferentiated ESCs, including Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.
o Telomerase Activity: Telomerases are necessary to sustain cell division unrestricted by the Hayflick limit of ~50 cell divisions. hESCs express high telomerase activity to sustain self-renewal and proliferation, and iPSCs also demonstrate high telomerase activity and express hTERT (human telomerase reverse transcriptase), a necessary component in the telomerase protein complex.
* Pluripotency: iPSCs were capable of differentiation in a fashion similar to ESCs into fully differentiated tissues.
o Neural Differentiation: iPSCs were differentiated into neurons, expressing βIII-tubulin, tyrosine hydroxylase, AADC, DAT, ChAT, LMX1B, and MAP2. The presence of catecholamine-associated enzymes may indicate that iPSCs, like hESCs, may be differentiable into dopaminergic neurons. Stem cell-associated genes were downregulated after differentiation.
o Cardiac Differentiation: iPSCs were differentiated into cardiomyocytes that spontaneously began beating. Cardiomyocytes expressed TnTc, MEF2C, MYL2A, MYHCβ, and NKX2.5. Stem cell-associated genes were downregulated after differentiation.
o Teratoma Formation: iPSCs injected into immunodeficient mice spontaneously formed teratomas after nine weeks. Teratomas are tumors of multiple lineages containing tissue derived from the three germ layers endoderm, mesoderm and ectoderm; this is unlike other tumors, which typically are of only one cell type. Teratoma formation is a landmark test for pluripotency.
o Embryoid Body: hESCs in culture spontaneously form ball-like embryo-like structures termed “embryoid bodies”, which consist of a core of mitotically active and differentiating hESCs and a periphery of fully differentiated cells from all three germ layers. iPSCs also form embryoid bodies and have peripheral differentiated cells.
o Blastocyst Injection: hESCs naturally reside within the inner cell mass (embryoblast) of blastocysts, and in the embryoblast, differentiate into the embryo while the blastocyst’s shell (trophoblast) differentiates into extraembryonic tissues. The hollow trophoblast is unable to form a living embryo, and thus it is necessary for the embryonic stem cells within the embryoblast to differentiate and form the embryo. iPSCs were injected by micropipette into a trophoblast, and the blastocyst was transferred to recipient females. Chimeric living mouse pups were created: mice with iPSC derivatives incorporated all across their bodies with 10%-90& chimerism.
* Epigenetic reprogramming
o Promoter Demethylation: Methylation is the transfer of a methyl group to a DNA base, typically the transfer of a methyl group to a cytosine molecule in a CpG site (adjacent cytosine/guanine sequence). Widespread methylation of a gene interferes with expression by preventing the activity of expression proteins or recruiting enzymes that interfere with expression. Thus, methylation of a gene effectively silences it by preventing transcription. Promoters of pluripotency-associated genes, including Oct-3/4, Rex1, and Nanog, were demethylated in iPSCs, demonstrating their promoter activity and the active promotion and expression of pluripotency-associated genes in iPSCs.
o Histone Demethylation: Histones are compacting proteins that are structurally localized to DNA sequences that can effect their activity through various chromatin-related modifications. H3 histones associated with Oct-3/4, Sox2, and Nanog were demethylated, indicating the expression of Oct-3/4, Sox2, and Nanog.
[ 本帖最后由 Tuexy 于 2009-2-2 20:46 编辑 ] |
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