Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming by these transcription factors is a continuous stochastic process where almost all mouse donor cells eventually give rise to iPS cells on continued growth and transcription factor expression. Additional inhibition of the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPS cell formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell-division-rate-independent manner. Quantitative analyses define distinct cell-division-rate-dependent and -independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency.

Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the postimplantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the nonobese diabetic (NOD) mouse strain, which prior to this study was considered "nonpermissive" for ESC derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ESCs and iPSCs appear to be "metastable," as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.

Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors. Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced. This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.

Runx2 is a master transcription factor for chondrocyte and osteoblast differentiation and bone formation. However expression of Runx2 (by RT-PCR), has been reported in non-skeletal tissues such as breast, T cells and testis. To better define Runx2 activity in non-skeletal tissues, we examined transgenic (Tg) mice expressing LacZ gene under control of 3.0 kb (3 kb Tg) or 1.0 kb (1 kb Tg) of the Runx2 distal (P1) promoter, Runx2 LacZ knock-in (Runx2(+/LacZ)) and Runx2/P1 LacZ knock-in (Runx2/P1(+/LacZ)). In the Runx2 3 kb Tg mouse, beta-galactosidase (beta-gal) expression appeared in various non-skeletal tissues including testis, skin, adrenal gland and brain. beta-gal expression from both 3 kb and 1 kb Tg, reflecting activity of the Runx2 promoter, was readily detectable in seminiferous tubules of the testis and the epididymis. At the single cell level, beta-gal was detected in spermatids and mature sperms not in sertoli or Leydig cells. We also detected a positive signal from the Runx2(+/LacZ) and Runx2/P1(+/LacZ) mice. Indeed, Runx2 expression was observed in isolated mature sperms, which was confirmed by RT-PCR and Western blot analysis. Runx2, however, was not related to sex determination and sperm motility. Runx2 mediated beta-gal activity is also found robustly in the hippocampus and frontal lobe of the brain in Runx2(+/LacZ). Collectively, these results indicate that Runx2 is expressed in several non-skeletal tissues particularly sperms of testis and hippocampus of brain. It suggests that Runx2 may play an important role in male reproductive organ testis and brain. J. Cell. Physiol.(c) 2008 Wiley-Liss, Inc.

The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency,(ii) the duration of transgene activity directly correlates with reprogramming efficiency,(iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.

Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.

Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming.

Since its discovery as a critical regulator of pluripotency in embryonic stem (ES) cells and the inner cells mass of the developing blastocyst, the Pou domaincontaining transcription factor Oct4 has become a proxy for "stemness" in numerous studies of somatic stem cells as it's presence is often taken as evidence of pluripotency in these cells. Recent studies, however, have demonstrated that not only is Oct4 dispensable for maintaining potency in somatic stem cell compartments, but also that the methods applied to detect Oct4 and the interpretation of the resulting data may be flawed. Here we contrast pathways known to govern pluripotency in embryonic stem cells with those in adult stem cells and critically discuss the concept of pluripotency in adult stem cells of the mammalian soma.

The Pou domain containing transcription factor Oct4 is a well-established regulator of pluripotency in the inner cell mass of the mammalian blastocyst as well as in embryonic stem cells. While it has been shown that the Oct4 gene is inactivated through a series of epigenetic modifications following implantation, recent studies have detected Oct4 activity in a variety of somatic stem cells and tumor cells. Based on these observations it has been suggested that Oct4 may also function in maintaining self-renewal of somatic stem cells and, in addition, may promote tumor formation. We employed a genetic approach to determine whether Oct4 is important for maintaining pluripotency in the stem cell compartments of several somatic tissues including the intestinal epithelium, bone marrow (hematopoietic and mesenchymal lineages), hair follicle, brain, and liver. Oct4 gene ablation in these tissues revealed no abnormalities in homeostasis or regenerative capacity. We conclude that Oct4 is dispensable for both self-renewal and maintenance of somatic stem cells in the adult mammal.

We present an overview of the concepts of tissue-specific transcriptional control mechanisms essential for development of the bone cell phenotype. BMP2 induced transcription factors constitute a network of activities and molecular switches for bone development and osteoblast differentiation. Among these regulators are Runx2 (Cbfa1/AML3), the principal osteogenic master gene for bone formation, as well as homeodomain proteins and osterix. Runx2 has multiple regulatory activities, including activation or repression of gene expression, and integration of biological signals from developmental cues, such as BMP/TGFbeta, Wnt and Src signaling pathways. Runx2 provides a new paradigm for transcriptional control by functioning as a principal scaffolding protein in nuclear microenvironments to control gene expression in response to physiologic signals (growth factors, cytokines and hormones). The protein serves as a hub for the coordination of activities essential for the expansion and differentiation of osteogenic lineage cells through the formation of co-regulatory protein complexes organized in subnuclear domains. Mechanisms by which Runx2 supports commitment to osteogenesis and determines cell fate involve its retention on mitotic chromosomes. Disruption of a unique protein module, the subnuclear targeting signal of Runx2, has profound effects on osteoblast differentiation and metastasis of cancer cells in the bone microenvironment. Runx2 target genes include regulators of cell growth control, components of the bone extracellular matrix, angiogenesis, and signaling proteins for development of the osteoblast phenotype and bone turnover. The specificity of Runx2 regulatory activities provides a basis for novel therapeutic strategies to correct bone disorders.