Hongkui Deng

Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds

Promoting Pluripotency A specialized mammalian cell can be set back to the pluripotent state either by transfer of the somatic cell nucleus into an oocyte or by delivery of exogenous pluripotency-associated transcription factors. Hou et al. (p. 651, published online 18 July) developed an approach to induce pluripotency in somatic cells using a cocktail of small molecules. The ability to generate such chemically induced pluripotent stem cells may provide an alternate route for therapeutic cloning and for drug development in regenerative medicine. A proof-of-principle study reports somatic reprogramming to the pluripotent state using small-molecule compounds. Pluripotent stem cells can be induced from somatic cells, providing an unlimited cell resource, with potential for studying disease and use in regenerative medicine. However, genetic manipulation and technically challenging strategies such as nuclear transfer used in reprogramming limit their clinical applications. Here, we show that pluripotent stem cells can be generated from mouse somatic cells at a frequency up to 0.2% using a combination of seven small-molecule compounds. The chemically induced pluripotent stem cells resemble embryonic stem cells in terms of their gene expression profiles, epigenetic status, and potential for differentiation and germline transmission. By using small molecules, exogenous “master genes” are dispensable for cell fate reprogramming. This chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications.

Expression cloning of new receptors used by simian and human immunodeficiency viruses

Several members of the chemokine-receptor family serve, in conjunction with CD4, as receptors for the entry of human immunodeficiency virus type I (HIV-1) into cells. The principal receptor for entry of macrophage-tropic (M-tropic) HIV-1 strains is CCR5, whereas that for T-cell-line-tropic (T-tropic) strains is CXCR4. Unlike HIV-1, infection with either M-tropic or T-tropic strains of simian immunodeficiency virus (SIV) can be mediated by CCR5, but not CXCR4 (refs 7, 8, 9, 10). SIV strains will also infect CD4+ cells that lack CCR5, which suggests that these strains use as yet unidentified receptors. Here we use an expression-cloning strategy to identify SIV receptors and have isolated genes encoding two members of the seven-transmembrane G-protein-coupled receptor family that are used not only by SIVs, but also by strains of HIV-2 and M-tropic HIV-1. Both receptors are closely related to the chemokine-receptor family and are expressed in lymphoid tissues. One of the receptors is also expressed in colon and may therefore be important in viral transmission. Usage of these new receptors following experimental infection of non-human primates with SIV strains may provide important insight into viral transmission and the mechanisms of SIV- and HIV-induced acquired immune-deficiency syndrome.

Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.

(Cell Stem Cell 4, 16–19; January 9, 2009)In our recent article, we unfortunately misquoted the findings in a recent study by Ying et al. (2008)xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al. (2008). When describing previous work using combination of the MEK inhibitor PD0325901 and the GSK3b inhibitor CHIR99021, our statement “Recent studies demonstrated that addition of the FGFR inhibitor PD173074 to the above cocktail is sufficient to maintain mESC pluripotency in the absence of LIF (Ying et al., 2008xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al., 2008).” was not accurate. Ying et al., in fact, used PD0325901 to replace PD1730474 and demonstrated maintenance of mESCs in the absence of LIF with these two factors only.The corrected section reads “Indeed, after serial passages, the growth of putative riPSCs treated with only PD0325901 and CHIR99021 declined, and the culture deteriorated due to the expansion of differentiated cells, although recent studies demonstrated that this combination of inhibitors can be used to maintain mESC self-renewal in the absence of LIF (Ying et al., 2008xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al., 2008). Because the TGFβ/Activin A/Nodal signaling cascade is essential to maintain undifferentiated hESCs and EpiSCs, but dispensable for mESC self-renewal, we tested whether the addition of an inhibitor of the type 1 TGFβ receptor, ALK5 (A-83-01), could help stabilize our riPSC cultures.”In addition, we omitted to mention that while our study was under review, Silva et al. (2008)xPromotion of Reprogramming to Ground State Pluripotency by Signal Inhibition. Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T.W., and Smith, A. PLoS Biol. 2008; 6: 2237–2247Crossref | Scopus (456)See all ReferencesSilva et al. (2008) also published the use of these two inhibitors in the generation and propagation of mouse iPSCs.We apologize for any confusion caused.

Directed differentiation of human embryonic stem cells into functional hepatic cells.

The differentiation capacity of human embryonic stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three‐stage method to efficiently direct the differentiation of human embryonic stem cells into hepatic cells in serum‐free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next, the presence of fibroblast growth factor‐4 and bone morphogenetic protein‐2 in the culture medium for 5 days induced efficient hepatic differentiation from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After 10 days of further in vitro maturation, these cells expressed the adult liver cell markers tyrosine aminotransferase, tryptophan oxygenase 2, phosphoenolpyruvate carboxykinase (PEPCK), Cyp7A1, Cyp3A4 and Cyp2B6. Furthermore, these cells exhibited functions associated with mature hepatocytes including albumin secretion, glycogen storage, indocyanine green, and low‐density lipoprotein uptake, and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice, these cells integrated into the mouse liver and expressed human alpha‐1 antitrypsin for at least 2 months. In addition, we found that the hESC‐derived hepatic cells were readily infected by human immunodeficiency virus‐hepatitis C virus pseudotype viruses. Conclusion: We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development, and form the basis for hepatocyte transplantation and drug tests. (HEPATOLOGY 2007;45:1229–1239.)

Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells

Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDX1-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insulin-producing cells. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.

Generation of Induced Pluripotent Stem Cells from Adult Rhesus Monkey Fibroblasts

Induced pluripotent stem (iPS) cells can be generated from somatic cells by transduction with several transcription factors in mouse and human. However, direct reprogramming in other species has not been reported. Here, we generated monkey iPS cells by retrovirus-mediated introduction of monkey transcription factors OCT4, SOX2, KLF4, and c-MYC.

Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells

Human induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells, and can proliferate intensively and differentiate into a variety of cell types. However, the hepatic differentiation of human iPS cells has not yet been reported. In this report, human iPS cells were induced to differentiate into hepatic cells by a stepwise protocol. The expression of liver cell markers and liver-related functions of the human iPS cell-derived cells were monitored and compared with that of differentiated human ES cells and primary human hepatocytes. Approximately 60% of the differentiated human iPS cells at day 7 expressed hepatic markers alpha fetoprotein and Alb. The differentiated cells at day 21 exhibited liver cell functions including albumin Asecretion, glycogen synthesis, urea production and inducible cytochrome P450 activity. The expression of hepatic markers and liver-related functions of the iPS cell-derived hepatic cells were comparable to that of the human ES cell-derived hepatic cells. These results show that human iPS cells, which are similar to human ES cells, can be efficiently induced to differentiate into hepatocyte-like cells.

In vitro derivation of functional insulin-producing cells from human embryonic stem cells

The capacity for self-renewal and differentiation of human embryonic stem (ES) cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid (RA) was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdx1 and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin (STZ)-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdx1, glucokinase, nkx6.1, IAPP, pax6 and Tcf1. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.

Generation of iPSCs from mouse fibroblasts with a single gene, Oct4 , and small molecules

The introduction of four transcription factors Oct4, Klf4, Sox2 and c-Myc by viral transduction can induce reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), but the use of iPSCs is hindered by the use of viral delivery systems. Chemical-induced reprogramming offers a novel approach to generating iPSCs without any viral vector-based genetic modification. Previous reports showed that several small molecules could replace some of the reprogramming factors although at least two transcription factors, Oct4 and Klf4, are still required to generate iPSCs from mouse embryonic fibroblasts. Here, we identify a specific chemical combination, which is sufficient to permit reprogramming from mouse embryonic and adult fibroblasts in the presence of a single transcription factor, Oct4, within 20 days, replacing Sox2, Klf4 and c-Myc. The iPSCs generated using this treatment resembled mouse embryonic stem cells in terms of global gene expression profile, epigenetic status and pluripotency both in vitro and in vivo. We also found that 8 days of Oct4 induction was sufficient to enable Oct4-induced reprogramming in the presence of the small molecules, which suggests that reprogramming was initiated within the first 8 days and was independent of continuous exogenous Oct4 expression. These discoveries will aid in the future generation of iPSCs without genetic modification, as well as elucidating the molecular mechanisms that underlie the reprogramming process.

Regulation of Apoptosis and Differentiation by p53 in Human Embryonic Stem Cells

The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs), hESCs normally undergo high rates of spontaneous apoptosis and differentiation, making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However, despite the accumulation of p53, it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types, including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation.