William E. Lowry

Generation of human induced pluripotent stem cells from dermal fibroblasts.

The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ectopic expression of the defined transcription factors KLF4, OCT4, SOX2, and C-MYC. The resultant cell lines are morphologically indistinguishable from human embryonic stem cells (HESC) generated from the inner cell mass of a human preimplantation embryo. Consistent with these observations, human iPS cells share a nearly identical gene-expression profile with two established HESC lines. Importantly, DNA fingerprinting indicates that the human iPS cells were derived from the donor material and are not a result of contamination. Karyotypic analyses demonstrate that reprogramming of human cells by defined factors does not induce, or require, chromosomal abnormalities. Finally, we provide evidence that human iPS cells can be induced to differentiate along lineages representative of the three embryonic germ layers indicating the pluripotency of these cells. Our findings are an important step toward manipulating somatic human cells to generate an unlimited supply of patient-specific pluripotent stem cells. In the future, the use of defined factors to change cell fate may be the key to routine nuclear reprogramming of human somatic cells.

Induced Pluripotent Stem Cells and Embryonic Stem Cells Are Distinguished by Gene Expression Signatures

Induced pluripotent stem cells (iPSCs) outwardly appear to be indistinguishable from embryonic stem cells (ESCs). A study of gene expression profiles of mouse and human ESCs and iPSCs suggests that, while iPSCs are quite similar to their embryonic counterparts, a recurrent gene expression signature appears in iPSCs regardless of their origin or the method by which they were generated. Upon extended culture, hiPSCs adopt a gene expression profile more similar to hESCs; however, they still retain a gene expression signature unique from hESCs that extends to miRNA expression. Genome-wide data suggested that the iPSC signature gene expression differences are due to differential promoter binding by the reprogramming factors. High-resolution array profiling demonstrated that there is no common specific subkaryotypic alteration that is required for reprogramming and that reprogramming does not lead to genomic instability. Together, these data suggest that iPSCs should be considered a unique subtype of pluripotent cell.

Identification and Classification of Chromosomal Aberrations in Human Induced Pluripotent Stem Cells

Because of their somatic cell origin, human induced pluripotent stem cells (HiPSCs) are assumed to carry a normal diploid genome, and adaptive chromosomal aberrations have not been fully evaluated. Here, we analyzed the chromosomal integrity of 66 HiPSC and 38 human embryonic stem cell (HESC) samples from 18 different studies by global gene expression meta-analysis. We report identification of a substantial number of cell lines carrying full and partial chromosomal aberrations, half of which were validated at the DNA level. Several aberrations resulted from culture adaptation, and others are suspected to originate from the parent somatic cell. Our classification revealed a third type of aneuploidy already evident in early passage HiPSCs, suggesting considerable selective pressure during the reprogramming process. The analysis indicated high incidence of chromosome 12 duplications, resulting in significant enrichment for cell cycle-related genes. Such aneuploidy may limit the differentiation capacity and increase the tumorigenicity of HiPSCs.

Src Tyrosine Kinase Is a Novel Direct Effector of G Proteins

Heterotrimeric G proteins transduce signals from cell surface receptors to modulate the activity of cellular effectors. Src, the product of the first characterized proto-oncogene and the first identified protein tyrosine kinase, plays a critical role in the signal transduction of G protein-coupled receptors. However, the mechanism of biochemical regulation of Src by G proteins is not known. Here we demonstrate that Galphas and Galphai, but neither Galphaq, Galpha12 nor Gbetay, directly stimulate the kinase activity of downregulated c-Src. Galphas and Galphai similarly modulate Hck, another member of Src-family tyrosine kinases. Galphas and Galphai bind to the catalytic domain and change the conformation of Src, leading to increased accessibility of the active site to substrates. These data demonstrate that the Src family tyrosine kinases are direct effectors of G proteins.

Structural Basis for Relief of Autoinhibition of the Dbl Homology Domain of Proto-Oncogene Vav by Tyrosine Phosphorylation

Rho-family GTPases transduce signals from receptors leading to changes in cell shape and motility, mitogenesis, and development. Proteins containing the Dbl homology (DH) domain are responsible for activating Rho GTPases by catalyzing the exchange of GDP for GTP. Receptor-initiated stimulation of Dbl protein Vav exchange activity involves tyrosine phosphorylation. We show through structure determination that the mVav1 DH domain is autoinhibited by an N-terminal extension, which lies in the GTPase interaction site. This extension contains the Tyr174 Src-family kinase recognition site, and phosphorylation or truncation of this peptide results in stimulation of GEF activity. NMR spectroscopy data show that the N-terminal peptide is released from the DH domain and becomes unstructured upon phosphorylation. Thus, tyrosine phosphorylation relieves autoinhibition by exposing the GTPase interaction surface of the DH domain, which is obligatory for Vav activation.

Directed differentiation of human induced pluripotent stem cells generates active motor neurons

The potential for directed differentiation of human‐induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS‐derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS‐derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS‐derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases. STEM CELLS 2009;27:806–811

Progress in understanding reprogramming to the induced pluripotent state

Induction of pluripotency by transcription factors has become a commonplace method to produce pluripotent stem cells. Great strides have been made in our understanding of the mechanism by which this occurs — particularly in terms of transcriptional and chromatin-based events — yet only a small part of the complete picture has been revealed. Understanding the mechanism of reprogramming to pluripotency will have important implications for improving the efficiency and quality of reprogramming and advancing therapeutic application of induced pluripotent stem cells. It will also help to reveal the machinery that stabilizes cell identity and to instruct the design of directed differentiation or lineage switching strategies. To inform the next phase in understanding reprogramming, we review the latest findings, highlight ongoing debates and outline future challenges.

Female Human iPSCs Retain an Inactive X Chromosome

Generating induced pluripotent stem cells (iPSCs) requires massive epigenome reorganization. It is unclear whether reprogramming of female human cells reactivates the inactive X chromosome (Xi), as in mouse. Here we establish that human (h)iPSCs derived from several female fibroblasts under standard culture conditions carry an Xi. Despite the lack of reactivation, the Xi undergoes defined chromatin changes, and expansion of hiPSCs can lead to partial loss of XIST RNA. These results indicate that hiPSCs are epigenetically dynamic and do not display a pristine state of X inactivation with two active Xs as found in some female human embryonic stem cell lines. Furthermore, whereas fibroblasts are mosaic for the Xi, hiPSCs are clonal. This nonrandom pattern of X chromosome inactivation in female hiPSCs, which is maintained upon differentiation, has critical implications for clinical applications and disease modeling, and could be exploited for a unique form of gene therapy for X-linked diseases.

Derivation of Primordial Germ Cells from Human Embryonic and Induced Pluripotent Stem Cells Is Significantly Improved by Coculture with Human Fetal Gonadal Cells

The derivation of germ cells from human embryonic stem cells (hESCs) or human induced pluripotent stem (hIPS) cells represents a desirable experimental model and potential strategy for treating infertility. In the current study, we developed a triple biomarker assay for identifying and isolating human primordial germ cells (PGCs) by first evaluating human PGC formation during the first trimester in vivo. Next, we applied this technology to characterizing in vitro derived PGCs (iPGCs) from pluripotent cells. Our results show that codifferentiation of hESCs on human fetal gonadal stromal cells significantly improves the efficiency of generating iPGCs. Furthermore, the efficiency was comparable between various pluripotent cell lines regardless of origin from the inner cell mass of human blastocysts (hESCs), or reprogramming of human skin fibroblasts (hIPS). To better characterize the iPGCs, we performed Real‐time polymerase chain reaction, microarray, and bisulfite sequencing. Our results show that iPGCs at day 7 of differentiation are transcriptionally distinct from the somatic cells, expressing genes associated with pluripotency and germ cell development while repressing genes associated with somatic differentiation (specifically multiple HOX genes). Using bisulfite sequencing, we show that iPGCs initiate imprint erasure from differentially methylated imprinted regions by day 7 of differentiation. However, iPGCs derived from hIPS cells do not initiate imprint erasure as efficiently. In conclusion, our results indicate that triple positive iPGCs derived from pluripotent cells differentiated on hFGS cells correspond to committed first trimester germ cells (before 9 weeks) that have initiated the process of imprint erasure. STEM CELLS 2009;27:783–795

Molecular Analyses of Human Induced Pluripotent Stem Cells and Embryonic Stem Cells

Recent work from our group and others has argued that human induced pluripotent stem cells (hiPSCs) generated by the introduction of four viruses bearing reprogramming factors differ from human embryonic stem cells (hESCs) at the level of gene expression (Chin et al., 2009). Many of the differences seen were common across independent labs and, at least to some extent, are thought to be a result of residual expression of donor cell-specific genes (Chin et al., 2009; Ghosh et al., 2010; Marchetto et al., 2009). Two new reports reanalyze similar expression data sets as those used in Chin et al. (2009) and come to different conclusions (Newman and Cooper, 2010; Guenther et al., 2010). We compare various approaches to perform gene expression meta-analysis that all support our original conclusions and present new data to demonstrate that polycistronic delivery of the reprogramming factors and extended culture brings hiPSCs transcriptionally closer to hESCs.