Weiqi Zhang

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.

Progressive degeneration of human neural stem cells caused by pathogenic LRRK2

Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing. It is therefore plausible that diseases whose manifestations correlate with ageing might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of ageing-associated disorders by focusing on a leucine-rich repeat kinase 2 (LRRK2) dominant mutation (G2019S; glycine-to-serine substitution at amino acid 2019), which is associated with familial and sporadic Parkinson’s disease as well as impairment of adult neurogenesis in mice. Here we report on the generation of induced pluripotent stem cells (iPSCs) derived from Parkinson’s disease patients and the implications of LRRK2(G2019S) mutation in human neural-stem-cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in nuclear-envelope organization, clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in Parkinson’s disease iPSCs and were recapitulated after targeted knock-in of the LRRK2(G2019S) mutation in human embryonic stem cells. Analysis of human brain tissue showed nuclear-envelope impairment in clinically diagnosed Parkinson’s disease patients. Together, our results identify the nucleus as a previously unknown cellular organelle in Parkinson’s disease pathology and may help to open new avenues for Parkinson’s disease diagnoses as well as for the potential development of therapeutics targeting this fundamental cell structure.

Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging.

Heterochromatin in aging stem cells Analysis of human aging syndromes, such as Werner syndrome (WS), may lead to greater understanding of both premature and normal aging. Zhang et al. generated isogenic WS-specific human embryonic stem cell lines (see the Perspective by Brunauer and Kennedy). WS-mesenchymal stem cells displayed features characteristic of premature aging, including heterochromatin disorganization. WRN protein thus functions in the maintenance of heterochromatin, and heterochromatin alterations may represent a driving force of human aging. Science, this issue p. 1160; see also p. 1093 Stabilization of heterochromatin by WRN protein safeguards human mesenchymal stem cells from aging. [Also see Perspective by Brunauer and Kennedy] Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1α and nuclear lamina–heterochromatin anchoring protein LAP2β. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging.

SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2

SIRT6 belongs to the mammalian homologs of Sir2 histone NAD+-dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout human mesenchymal stem cells (hMSCs) by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated functional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sensitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 (NRF2) and RNA polymerase II, which was required for the transactivation of NRF2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Overexpression of HO-1 in SIRT6-null hMSCs rescued premature cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay.

Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs

Fanconi anaemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow (BM) failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration free-induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA-deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA patient BM cells.

Switching cell fate, ncRNAs coming to play

Cell fate decision is a critical step during physiological development when embryonic stem cells commit to either becoming adult stem cells or somatic cells. Recent advances in reprogramming demonstrate that a similar set of transcription factors (TFs), which are important for maintaining the pluripotent state of stem cells, can also reprogram somatic cells to induced pluripotent stem cells (iPSCs). In addition, trans-differentiation, which entails the use of different sets of defined factors, whereby one type of somatic cell can be directly converted into another and even to cell types from different germ layers has become a parallel widely used approach for switching cell fate. All these progresses have provided powerful tools to manipulate cells for basic science and therapeutic purposes. Besides protein-based factors, non-coding RNAs (ncRNAs), particularly microRNAs and long ncRNAs, are also involved in cell fate determination, including maintaining self-renewal of pluripotent stem cells and directing cell lineage. Targeting specific ncRNAs represents an alternative promising approach to optimize cell-based disease modeling and regenerative therapy. Here we focus on recent advances of ncRNAs in cell fate decision, including ncRNA-induced iPSCs and lineage conversion. We also discuss some underlying mechanisms and implications in molecular pathogenesis of human diseases.

Dual induction of apoptotic and autophagic cell death by targeting survivin in head neck squamous cell carcinoma.

Survivin is ubiquitously expressed in patients with head neck squamous cell carcinoma (HNSCC) and is associated with poor survival and chemotherapy resistance. Sepantronium bromide (YM155) is a selective survivin suppressant that exhibits potent antitumor activities by inducing apoptosis and autophagy in various types of cancer. However, the curative effects and underlying mechanisms of YM155 in HNSCC remain unclear. This study showed that survivin overexpression positively correlated with p-S6, p-Rb and LAMP2 but negatively correlated with the autophagic marker LC3 in human HNSCC tissues. In vitro studies revealed that YM155 triggered apoptosis of HNSCC cells in mitochondria and death receptor-dependent manner. The treatment also significantly enhanced autophagy by upregulating Beclin1, which led to cell death. YM155 not only downregulated the expression of survivin but also remarkably suppressed the activation of the mTOR signaling pathway in vitro and in vivo. YM155 displayed potent antitumor activities in both CAL27 xenograft and transgenic HNSCC mice models by delaying tumor onset and suppressing tumor growth. Furthermore, YM155 combined with docetaxel promoted tumor regression better than either treatment alone without causing considerable body weight loss in the HNSCC xenograft models. Overall, targeting survivin by YM155 can benefit HNSCC therapy by increasing apoptotic and autophagic cell death, and suppressing prosurvival pathways.

Find and replace: editing human genome in pluripotent stem cells

Genetic manipulation of human pluripotent stem cells (hPSCs) provides a powerful tool for modeling diseases and developing future medicine. Recently a number of independent genome-editing techniques were developed, including plasmid, bacterial artificial chromosome, adeno-associated virus vector, zinc finger nuclease, transcription activator-like effecter nuclease, and helper-dependent adenoviral vector. Gene editing has been successfully employed in different aspects of stem cell research such as gene correction, mutation knock-in, and establishment of reporter cell lines (Raya et al., 2009; Howden et al., 2011; Li et al., 2011; Liu et al., 2011b; Papapetrou et al., 2011; Sebastiano et al., 2011; Soldner et al., 2011; Zou et al., 2011a). These techniques combined with the utility of hPSCs will significantly influence the area of regenerative medicine.

Testes-specific protease 50 promotes cell invasion and metastasis by increasing NF-kappaB-dependent matrix metalloproteinase-9 expression

The high mortality in breast cancer is often associated with metastatic progression in patients. Previously we have demonstrated that testes-specific protease 50 (TSP50), an oncogene overexpressed in breast cancer samples, could promote cell proliferation and tumorigenesis. However, whether TSP50 also has a key role in cell invasion and cancer metastasis, and the mechanism underlying the process are still unclear. Here we found that TSP50 overexpression greatly promoted cell migration, invasion, adhesion and formation of the stellate structures in 3D culture system in vitro as well as lung metastasis in vivo. Conversely, TSP50 knockdown caused the opposite changes. Mechanistic studies revealed that NF-κB signaling pathway was required for TSP50-induced cell migration and metastasis, and further results indicated that TSP50 overexpression enhanced expression and secretion of MMP9, a target gene of NF-κB signaling. In addition, knockdown of MMP9 resulted in inhibition of cell migration and invasion in vitro and lung metastasis in vivo. Most importantly, immunohistochemical staining of human breast cancer samples strongly showed that the coexpression of TSP50 and p65 as well as TSP50 and MMP9 were correlated with increased metastasis and poor survival. Furthermore, we found that some breast cancer diagnosis-associated features such as tumor size, tumor grade, estrogen receptors (ER) and progesterone receptors (PR) levels, were correlated well with TSP50/p65 and TSP50/MMP9 expression status. Taken together, this work identified the TSP50 activation of MMP9 as a novel signaling mechanism underlying human breast cancer invasion and metastasis.

P21cip-Overexpression in the Mouse β Cells Leads to the Improved Recovery from Streptozotocin-Induced Diabetes

Under normal conditions, the regeneration of mouse β cells is mainly dependent on their own duplication. Although there is evidence that pancreatic progenitor cells exist around duct, whether non-β cells in the islet could also potentially contribute to β cell regeneration in vivo is still controversial. Here, we developed a novel transgenic mouse model to study the pancreatic β cell regeneration, which could specifically inhibit β cell proliferation by overexpressing p21 cip in β cells via regulation of the Tet-on system. We discovered that p21 overexpression could inhibit β-cell duplication in the transgenic mice and these mice would gradually suffer from hyperglycemia. Importantly, the recovery efficiency of the p21-overexpressing mice from streptozotocin-induced diabetes was significantly higher than control mice, which is embodied by better physiological quality and earlier emergence of insulin expressing cells. Furthermore, in the islets of these streptozotocin-treated transgenic mice, we found a large population of proliferating cells which expressed pancreatic duodenal homeobox 1 (PDX1) but not markers of terminally differentiated cells. Transcription factors characteristic of early pancreatic development, such as Nkx2.2 and NeuroD1, and pancreatic progenitor markers, such as Ngn3 and c-Met, could also be detected in these islets. Thus, our work showed for the first time that when β cell self-duplication is repressed by p21 overexpression, the markers for embryonic pancreatic progenitor cells could be detected in islets, which might contribute to the recovery of these transgenic mice from streptozotocin-induced diabetes. These discoveries could be important for exploring new diabetes therapies that directly promote the regeneration of pancreatic progenitors to differentiate into islet β cells in vivo.