Duanqing Pei

A Mesenchymal-to-Epithelial Transition Initiates and Is Required for the Nuclear Reprogramming of Mouse Fibroblasts

Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-beta1 and TGF-beta receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu. PAPERCLIP:

Vitamin C Enhances the Generation of Mouse and Human Induced Pluripotent Stem Cells

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However, the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound, vitamin C (Vc), enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence, a recently identified roadblock for reprogramming. In addition, Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process.

Genomic instability in laminopathy-based premature aging

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24−/− mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24−/− MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.

Transmembrane-deletion Mutants of the Membrane-type Matrix Metalloproteinase-1 Process Progelatinase A and Express Intrinsic Matrix-degrading Activity

Membrane-type matrix metalloproteinase-1 (MT-MMP-1) has been proposed to play a critical role in regulating the expression of tissue-invasive phenotypes in normal and neoplastic cells by directly or indirectly mediating the activation of progelatinase A. To begin characterizing MT-MMP-1 structure-function relationships, transmembrane-deletion mutants were constructed, and the processing of the zymogens as well as the enzymic activity of the mature proteinases was analyzed. We now demonstrate that pro-MT-MMP-1 mutants are efficiently processed to active proteinases following post-translational endoproteolysis immediately downstream of an Arg-Arg-Lys-Arg basic motif by a proprotein convertase-dependent pathway. The secreted form of active MT-MMP-1 not only displays an N terminus identical with that described for the processed wild-type enzyme at Tyr (Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grants, G. A., and Goldberg, G. I.(1995) J. Biol. Chem. 270, 5331-5338), but also directly mediated progelatinase A activation via a two-step proteolytic cascade indistinguishable from that observed with intact cells. Furthermore, although the only function previously ascribed to MT-MMP-1 is its ability to act as a progelatinase A activator, purified transmembrane deletion mutants also expressed proteolytic activities against a wide range of extracellular matrix molecules. Given recent reports that MT-MMP-1 ectodomains may undergo intercellular transfer in vivo (Okada, A., Bellocq, J.-P., Rouyer, N., Chenard, M.-P., Rio, M.-C., Chambon, P., and Basset, P.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2730-2734), our data suggest that soluble forms of the proteinase confer recipient cells with the ability to not only process progelatinase A, but also directly degrade extracellular matrix components.

Generation of induced pluripotent stem cell lines from tibetan miniature pig

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.

H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs

The induction of pluripotent stem cells (iPSCs) by defined factors is poorly understood stepwise. Here, we show that histone H3 lysine 9 (H3K9) methylation is the primary epigenetic determinant for the intermediate pre-iPSC state, and its removal leads to fully reprogrammed iPSCs. We generated a panel of stable pre-iPSCs that exhibit pluripotent properties but do not activate the core pluripotency network, although they remain sensitive to vitamin C for conversion into iPSCs. Bone morphogenetic proteins (BMPs) were subsequently identified in serum as critical signaling molecules in arresting reprogramming at the pre-iPSC state. Mechanistically, we identified H3K9 methyltransferases as downstream targets of BMPs and showed that they function with their corresponding demethylases as the on/off switch for the pre-iPSC fate by regulating H3K9 methylation status at the core pluripotency loci. Our results not only establish pre-iPSCs as an epigenetically stable signpost along the reprogramming road map, but they also provide mechanistic insights into the epigenetic reprogramming of cell fate.

The Histone Demethylases Jhdm1a/1b Enhance Somatic Cell Reprogramming in a Vitamin-C-Dependent Manner

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process, but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b, two known vitamin-C-dependent H3K36 demethylases, as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore, we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367, an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming.

Stem cell pluripotency and transcription factor Oct4

ABSTRACTMammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 21st has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarize the structure and function of Oct4 and address issues related to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cells.

Identification and Characterization of the Fifth Membrane-type Matrix Metalloproteinase MT5-MMP

A new member of the membrane-type matrix metalloproteinase (MT-MMP) subfamily tentatively named MT5-MMP was isolated from mouse brain cDNA library. It is predicted to contain (i) a candidate signal sequence, (ii) a propeptide region with the highly conserved PRCGVPD sequence, (iii) a potential furin recognition motif RRRRNKR, (iv) a zinc-binding catalytic domain, (v) a hemopexin-like domain, (vi) a 24-residue hydrophobic domain as a potential transmembrane domain, and (vii) a short cytosolic domain. Reverse transcriptase-polymerase chain reaction analysis of its transcripts indicates that MT5-MMP is expressed in a brain-specific manner consistent with the origin of its EST clone from cerebellum. It is also highly expressed during embryonic development at stages day 11 and 15. Like other MT-MMPs, MT5-MMP specifically activates progelatinase A when co-expressed in Madin-Darby canine kidney cells. Its ability to activate progelatinase A is dependent on its proteolytic activity since a mutation converting Glu to Ala in the zinc binding motif HE255LGH renders MT5-MMP inactive against progelatinase A. In contrast to other MT-MMPs, MT5-MMP tends to shed from cell surface as soluble proteinases, thus offering flexibility as both a cell bound and soluble proteinase for extracellular matrix remodeling processes. Taken together, these properties serve to distinguish MT5-MMP as a versatile MT-MMP playing an important role in extracellular matrix remodeling events in the brain and during embryonic development.

Generation of human induced pluripotent stem cells from urine samples

Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet, acquiring donor cells is, in most instances, an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances, as the isolation of urinary cells is simple (30 ml of urine are sufficient), cost-effective and universal (can be applied to any age, gender and race). Moreover, the entire procedure is reasonably quick—around 2 weeks for the urinary cell culture and 3–4 weeks for the reprogramming—and the yield of iPSC colonies is generally high—up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential, and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases, including those affecting the kidney.