Mesenchymal stem cells (MSCs) have the capacity for multilineage differentiation and are being explored as a source for stem cell-based therapies. Previous studies have shown that adhesion to extracellular matrix plays a critical role in guiding MSC differentiation to distinct lineages. Here we conducted a focused screen of microRNAs to reveal one microRNA, miR-125b, whose expression changes as a function of cell adhesion. miR-125b expression was upregulated by limiting cell-matrix adhesion using micropatterned substrates, knocking down beta5 integrin, or placing cells in suspension culture. Interestingly, we noted that suspending hMSCs did not induce substantial apoptosis (anoikis) as is typically observed in adherent cells. Although miR-125b appeared to have some effects of on hMSC differentiation, we demonstrated a striking role for miR-125b in protecting hMSCs from anoikis. Knockdown of miR-125b increased anoikis while expressing a mimic protected cells. Mechanistic studies demonstrated that miR-125b protected against anoikis by increasing ERK phosphorylation and by suppressing p53. Lastly, we found that miR-125b expression is quite limited in endothelial cells and MEFs. The rapid anoikis normally observed in these cells is antagonized by expressing a miR-125b mimic, and induced pluripotent stem (iPS) cells generated from the MEFs led to upregulated miR-125b expression. Together, these observations demonstrate a novel link between cell-matrix adhesion, miR-125b expression, and a stem-cell specific survival program triggered in adhesion-limited contexts such as might occur in early development and wound healing.

Interferon gamma (IFNγ) has been reported to induce osteoblastogenesis from mesenchymal stem cells (MSC) both in vitro and in vivo. With ageing, adipocytes outnumber osteoblasts within the bone microenvironment leading to a decrease in bone formation. Since both osteoblasts and adipocytes are of mesenchymal origin we hypothesized that IFNγ treatment might negatively affect adipogenesis while stimulates osteoblastogenesis in human MSC. To test this hypothesis, human MSC were induced to differentiate into adipocytes in the presence or absence of osteogenic doses of IFNγ (1, 10, 100 ng/ml). IFNγ-treated MSC showed a decrease in adipocyte differentiation and lipid deposition as compared with vehicle-treated controls. Additionally, adipogenic markers were significantly decreased by IFNγ treatment at the same doses that have been reported to have a strong osteogenic effect in vitro. Furthermore, DNA binding of PPARγ was significantly lower in IFNγ-treated differentiating MSC. Subsequently, ovariectomized C57BL6 mice were treated with osteogenic doses of IFNγ three times a week for 6 weeks. In distal femur, treated mice showed significantly higher hematopoiesis concomitant with lower levels of fat volume/total volume, adipocyte number and expression of adipogenic markers as compared with the vehicle-treated mice. Together, these findings demonstrate that, at osteogenic doses, IFNγ also acts as an inhibitor of adipogenesis in vitro and prevents marrow fat infiltration while favors hematopoiesis in ovariectomized mice.

Presence in glioblastomas of cancer cells with normal neural stem cell (NSC) properties, tumor initiating capacity, and resistance to current therapies, suggests that glioblastoma stem-like cells (GSCs) play central roles in glioblastoma development. We cultured human GSCs endowed with all features of tumor stem cells, including tumor initiation after xenograft and radio-chemoresistance. We established proteomes from four GSC cultures and their corresponding whole tumor tissues and from human NSCs. Two-dimensional difference gel electrophoresis and tandem mass spectrometry revealed a two-fold increase of Hepatoma-Derived Growth Factor (HDGF) in GSCs as compared to tumor tissues and NSCs. Western blot analysis confirmed HDGF over-expression in GSCs, as well as its presence in GSC-conditioned medium, while, in contrast, no HDGF was detected in NSC secretome. At the functional level, GSC-conditioned medium induced migration of human cerebral endothelial cells that can be blocked by anti-HDGF antibodies. In vivo, GSC-conditioned medium induced neo-angiogenesis, whereas HDGF-targeting siRNAs abrogated this effect. Altogether, our results identify a novel candidate, by which GSCs can support neo-angiogenesis, a high-grade glioma hallmark. Our strategy illustrates the usefulness of comparative proteomic analysis to decipher molecular pathways, which underlie GSC properties.

Label-retaining-cells (LRC) have been proposed to represent adult tissue stem-cells. LRC are hypothesized to result from either slow-cycling or asymmetric-cell-division. However, the stem-cell nature and whether LRC undergo asymmetric-cell-division remains controversial. Here we demonstrate label-retaining-cancer-cells (LRCC) in several gastrointestinal cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing, and exhibit stem-cells and pluripotency gene expression profiles. Using real-time confocal-microscopic-cinematography, we show live LRCC undergoing asymmetric non-random chromosomal cosegregation label-retaining cell division. Importantly, LRCC have greater tumor initiating capacity than non-LRCC. Based on our data and that cancers develop in tissues that harbor normal-LRC, we propose that LRCC might represent a novel population of gastrointestinal stem-like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine, and present novel targets for cancer treatment.

Despite burgeoning interest in the potential of cellular therapies in lung regenerative medicine, progress in delivering these therapies has been confounded by a lack of knowledge about the identity of appropriate targets which can be harnessed to repair the lung, and the cellular and molecular factors which regulate their regenerative potential. While systematic analysis of lung development, and cell lineage tracing studies in normal and perturbed animal models, provides a framework for understanding the complex interplay of the multiple cell types, biomatrix elements and soluble and insoluble cytokines and factors which regulate lung structure and function, a reductionist approach is also required to analyse the organisation of regenerative cells in the adult lung and identify the factors and molecular pathways which regulate their capacity to generate descendent lineages. In this review we describe recent progress in identifying and characterising endogenous epithelial, mesenchymal and endothelial stem/progenitor cells in the adult lung utilising multiparameter cell separative strategies and functional in vitro clonogenic assays.

The airways of the mammalian lung are lined with highly specialized epithelial cell types that are the target of airborne toxicants and injury. Notch signaling plays an important role in the ontogeny of airway epithelial cells, but its contributions to recruitment, expansion or differentiation of resident progenitor/stem cells and repair and reestablishment of the normal composition of airway epithelium following injury have not been addressed. In this study, the role of a specific Notch receptor, Notch1, was investigated by targeted inactivation in the embryonic lung epithelium using the epithelial-specific Gata5-Cre driver line. Notch1-deficient mice are viable without discernible defects in pulmonary epithelial cell fate determination and differentiation. However, in an experimental model of airway injury, activity of Notch1 is found to be required for normal repair of the airway epithelium. Absence of Notch1 reduced the ability of a population of cells distinguished by expression of PGP9.5, otherwise a marker of pulmonary neuroendocrine cells, which appears to serve as a reservoir for regeneration of Clara cells. Hairy/Enhancer of Split-5 (Hes5) and a paired-box-containing gene 6 (Pax6) were found to be downstream targets of Notch1. Both Hes5 and Pax6 expressions were significantly increased in association with Clara cell regeneration in wild type lungs. Ablation of Notch1 reduced Hes5 and Pax6 and inhibited airway epithelial repair. Thus, although dispensable in developmental ontogeny of airway epithelial cells, normal activity of Notch1 is required for repair of the airway epithelium. The signaling pathway by which Notch1 regulates the repair process includes stimulation of Hes5 and Pax6 gene expression.

For the safe clinical application of embryonic stem cells (ESCs) for neurological diseases, it is critical to evaluate the tumorigenicity and function of human ESC-derived neural cells in primates. We have herein, for the first time, compared the growth and function of human ESC-derived cells with different stages of neural differentiation implanted in the brains of primate models of Parkinson's disease. We herein show that residual undifferentiated cells expressing ESC markers present in the cell preparation can induce tumor formation in the monkey brain. In contrast, a cell preparation matured by 42-day-culture with BDNF/GDNF treatment did not form tumors, and survived as primarily dopaminergic neurons. In addition, the monkeys with such grafts showed behavioral improvement for at least 12 months. These results support the idea that human ESCs, if appropriately matured, can serve as a source for dopaminergic neurons without forming any tumors in a primate brain.

Breast cancers are thought to be organized hierarchically with a small number of breast cancer stem cells (BCSCs) able to re-grow a tumor while their progeny lack this ability. Recently, several groups reported enrichment for BCSCs when breast cancers were subjected to classical anticancer treatment. However, the underlying mechanisms leading to this enrichment are incompletely understood.

Using non-BCSCs sorted from patient samples, we found that ionizing radiation reprogrammed differentiated breast cancer cells into induced BCSCs (iBCSCs). iBCSCs showed increased mammosphere formation, increased tumorigenicity and expressed the same stemness-related genes as BCSCs from non-irradiated samples. Reprogramming occurred in a polyploid subpopulation of cells, coincided with re-expression of the transcription factors Oct4, Sox-2, Nanog, and Klf4, and could be partially prevented by Notch inhibition.

We conclude that radiation may induce a BCSC phenotype in differentiated breast cancer cells and that this mechanism contributes to increased BCSC numbers seen after classical anti-cancer treatment.

Mouse pluripotent stem cells (PSCs) are the best studied pluripotent system, and are regarded as the “gold standard” to which human PSCs are compared. However, while the genomic integrity of human PSCs has recently drawn much attention, mouse PSCs have not been systematically evaluated in this regard. The genomic stability of PSCs is a matter of profound significance, as it affects their pluripotency, differentiation and tumorigenicity. We thus performed a thorough analysis of the genomic integrity of 325 samples of mouse PSCs, including 127 induced pluripotent stem cell (iPSC) samples. We found that genomic aberrations occur frequently in mouse embryonic stem cells (ESCs) of various mouse strains, and in mouse iPSCs of various cell origins and derivation techniques. Four hotspots of chromosomal aberrations were detected: full trisomy 11 (with a minimally recurrent gain in 11qE2), full trisomy 8, and deletions in chromosomes 10qB and 14qC-14qE. The most recurrent aberration in mouse PSCs, gain 11qE2, turned out to be fully syntenic to the common aberration 17q25 in human PSCs, while other recurrent aberrations were surprisingly found to be species-specific. Analysis of chromosomal aberrations in 74 samples of rhesus macaque PSCs revealed a gain in chromosome 16q, syntenic to the hotspot in human 17q. Importantly, these common aberrations jeopardize the interpretation of published comparisons of PSCs, which were unintentionally conducted between normal and aberrant cells. Therefore, this work emphasizes the need to carefully monitor genomic integrity of PSCs from all species, for their proper use in biomedical research.

The invasive nature of glioblastoma is one important reason of treatment failure. Glioblastoma stem/progenitor cells retain the migratory ability of normal neural stem/progenitor cells and infiltrate the brain parenchyma. Here we identify Rai (ShcC/N-Shc), a member of the family of Shc-like adaptor proteins, as a new regulator of migration of normal and cancer stem/progenitor cells. Rai is expressed in neurogenic areas of the brain and its knock-down impairs progenitor migration to the olfactory bulb. Its expression is retained in glioblastoma stem/progenitor cells where it exerts the same promigratory activity. Rai-silencing in cancer stem/progenitor cells isolated from different patients causes significant decrease in cell migration and invasion, both in vitro and in vivo, providing survival benefit. Rai depletion is associated with alteration of multiple signaling pathways, yet it always leads to reduced expression of proinvasive genes.

Reprogramming of the somatic state to pluripotency can be induced by a defined set of transcription factors including Oct3/4, Sox2, Klf4 and c-Myc [1]. These induced pluripotent stem cells (iPSCs) hold great promise in human therapy and disease modeling. However, tumor suppressive activities of p53, which are necessary to prevent persistence of DNA damage in mammalian cells, have proven a serious impediment to formation of iPSCs [2]. We examined the requirement for downstream p53 activities in suppressing efficiency of reprogramming as well as preventing persistence of DNA damage into the early iPSCs. We discovered that the majority of the p53 activation occurred through early reprogramming-induced DNA damage with the activated expression of the apoptotic inducer Puma and the cell cycle inhibitor p21. While Puma-deficiency increases reprogramming efficiency only in the absence of c-Myc, double deficiency of Puma and p21 has achieved a level of efficiency that exceeded that of p53 deficiency alone. We further demonstrated that, in both the presence and absence of p21, Puma-deficiency was able to prevent any increase in persistent DNA damage in early iPSCs. This may be due to a compensatory cellular senescent-response to reprogramming-induced DNA damage in pre-iPSCs. Therefore, our findings provide a potentially safe approach to enhance iPSC derivation by transiently silencing Puma and p21 without compromising genomic integrity.

Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which NADPH oxidase is defective in generating reactive oxygen species. In this study, we reprogrammed three normal unrelated patient's fibroblasts (p47^phox and gp91^phox) to pluripotency by lentiviral transduction with defined pluripotency factors. These induced pluripotent stem cells (iPSC) share the morphological features of human embryonic stem cells, express the key pluripotency factors and posses high telomerase activity. Furthermore, all the iPSC lines formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo. They were isogenic with the original patient fibroblasts, exhibited normal karyotype and retained the p47^phox or gp91^phox mutations found in the patient fibroblasts. We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profile to blood-derived macrophages under resting conditions. Most importantly, CGD-patient specific iPSC derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients. Together these results suggest that CGD-patient-specific iPSC lines represent an important tool for modelling CGD disease phenotypes, screening candidate drugs and the development of gene therapy.

CD49f (integrin subunit alpha 6) regulates signaling pathways in a variety of cellular activities. However, the role of CD49f in regulating the differentiation and pluripotency of stem cells has not been fully investigated. Therefore, in this study, human mesenchymal stem cells (hMSCs) were induced to form spheres under non-adherent culture conditions, and we found that the CD49f-positive population was enriched in MSC spheres compared with MSCs in a monolayer. The expression of CD49f regulated the ability of hMSCs to form spheres and was associated with an activation of the PI3K/AKT signaling pathway. Furthermore, the forced expression of CD49f modulated the proliferation and differentiation potentials of hMSCs through prolonged activation of PI3K/AKT and suppressed the level of p53. We showed that the pluripotency factors OCT4 and SOX2 were recruited to the putative promoter region of CD49f, indicating that OCT4 and SOX2 play positive roles in the expression of CD49f. Indeed, CD49f expression was up-regulated in human embryonic stem cells (hESCs) compared with hMSCs. The elevated level of CD49f expression was significantly decreased upon embryoid body formation in hESCs. In hESCs, the knockdown of CD49f down-regulated PI3K/AKT signaling and up-regulated the level of p53, inducing differentiation into three germ layers. Taken together, our data suggest that the cell-surface protein CD49f has novel and dynamic roles in regulating the differentiation potential of hMSCs and maintaining pluripotency.

Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we employed a conventional lentiviral transduction method to derive human induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole body imaging technology, we demonstrated that after tail vein injection these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help overcome problems associated with allogeneic transplantation of other types of human NSCs.

RelB and NF-κB2 are the main effectors of NF-κB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-κB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-κB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment.

Bone marrow (BM) microenvironment, which is regulated by hypoxia and proteolytic enzymes, is crucial for stem/progenitor cell function and mobilization involved in postnatal neovascularization. We demonstrated that NADPH oxidase2 (Nox2)-derived reactive oxygen species (ROS) are involved in post-ischemic mobilization of BM cells and revascularization. However, role of Nox2 in regulating BM microenvironment in response to ischemic injury remains unknown. Here we show that hindlimb ischemia of mice increases ROS production in both the endosteal and central region of BM tissue in situ, which is almost completely abolished in Nox2 knockout (KO) mice. This Nox2-dependent ROS production is mainly derived from Gr-1⁺ myeloid cells in BM. In vivo injection of hypoxyprobe reveals that endosteum at the BM is hypoxic with high expression of HIF-1α in basal state. Following hindlimb ischemia, hypoxic areas and HIF-1α expression are expanded throughout the BM, which is inhibited in Nox2 KO mice. This ischemia-induced alteration of Nox2-dependent BM microenvironment is associated with an increase in VEGF expression and Akt phosphorylation in BM tissue, thereby promoting Lin^- progenitor cell survival and expansion, leading to their mobilization from BM. Furthermore, hindlimb ischemia increases proteolytic enzymes MT1-MMP expression and MMP-9 activity in BM, which is inhibited in Nox2 KO mice. In summary, Nox2-dependent increase in ROS play a critical role in regulating hypoxia expansion and proteolytic activities in BM microenvironment in response to tissue ischemia. This in turn promotes progenitor cell expansion and reparative mobilization from BM, leading to post-ischemic neovascularization and tissue repair.

Secreted Frizzled related proteins (sFRPs) are a family of proteins that modulate Wnt signaling, which in its turn regulates multiple aspects of ventral midbrain (VM) and dopaminergic (DA) neuron development. However, it is not known which Wnt signaling branch and what aspects of midbrain DA neuron development are regulated by sFRPs. Here, we show that sFRP1 and sFRP2 activate the Wnt/PCP/Rac1 pathway in DA cells. In the developing VM, sFRP1 and sFRP2 are expressed at low levels and sFRP1-/- or sFRP2-/- mice had no detectable phenotype. However, compound sFRP1-/-; sFRP2-/- mutants revealed a Wnt/PCP phenotype similar to that previously described for Wnt5a-/- mice. This included an antero-posterior shortening of the VM, a lateral expansion of the Shh domain and DA lineage markers (Lmx1a and Th), as well as an accumulation of Nurr1+ precursors in the VM. In vitro experiments showed that, while very high concentrations of SFRP1 had a negative effect on cell survival, low/medium concentrations of sFRP1 or sFRP2 promoted the DA differentiation of progenitors derived from primary VM cultures or mouse embryonic stem cells (mESC), mimicking the effects of Wnt5a. We thus conclude that the main function of sFRP1 and sFRP2 is to enhance Wnt/PCP signaling in DA cells and to regulate Wnt/PCP-dependent functions in midbrain development. Moreover, we suggest that low-medium concentrations of sFRPs may be used to enhance the DA differentiation of ES cells and improve their therapeutic application.

Background: Reno-protective strategies are needed to improve renal outcomes in patients with atherosclerotic renal artery stenosis (ARAS). Adipose tissue-derived mesenchymal stem cells (MSC) can promote renal regeneration, but their potential for attenuating cellular injury and restoring kidney repair in ARAS has not been explored. We hypothesized that replenishment of MSC as an adjunct to percutaneous transluminal renal angioplasty (PTRA) would restore renal cellular integrity and improve renal function in ARAS pigs.

Methods and Results: Four groups of pigs (n=7 each) were studied after 16 weeks of ARAS, ARAS 4 weeks after PTRA and stenting with or without adjunct intra-renal delivery of MSC (10x10(6) cells), and controls. Stenotic kidney blood flow (RBF) and glomerular filtration rate (GFR) were measured using multidetector CT. Renal microvascular architecture (micro-CT), fibrosis, inflammation, and oxidative stress were evaluated ex-vivo.

Four weeks after successful PTRA, mean arterial pressure fell to a similar level in all revascularized groups. Stenotic kidney GFR and RBF remained decreased in ARAS (p=0.01 and p=0.02) and ARAS+PTRA (p=0.02 and p=0.03) compared to normal, but rose to normal levels in ARAS+PTRA+MSC (p=0.34 and p=0.46 vs. normal). Interstitial fibrosis, inflammation, microvascular rarefaction, and oxidative stress were attenuated only in PTRA+MSC-treated pigs.

Conclusions: A single intra-renal delivery of MSC in conjunction with renal revascularization restored renal hemodynamics and function, and decreased inflammation, apoptosis, oxidative stress, microvascular loss, and fibrosis. This study suggests a unique and novel therapeutic potential for MSC in restoring renal function when combined with PTRA in chronic experimental renovascular disease.

The cytokine thrombopoietin (Tpo) plays a critical role in hematopoiesis by binding to the extracellular domain, and inducing homodimerization of the intracellular signaling domain of its receptor, cMpl. Mpl homodimerization can also be accomplished by binding of a synthetic ligand to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. Unexpectedly, in contrast to Tpo stimulation, robust erythropoiesis is induced after dimerization of F36VMpl in human CD34+ progenitor cells. The goal of this study was to define the hematopoietic progenitor stages at which dimerization of intracellular Mpl induces erythropoiesis, and the downstream molecular events that mediate this unanticipated effect. Dimerization (in the absence of erythropoietin and other cytokines) in human Common Myeloid Progenitors (CMP) and Megakaryocytic-Erythroid Progenitors (MEP) caused a significant increase in CD34+ cells (p<0.01) and induced all stages of erythropoiesis including production of enucleated red blood cells. In contrast, erythropoiesis was not seen with Tpo stimulation. CD34+ cell expansion was the result of increased cell cycling and survival (p<0.05). Microarray profiling of CD34+ cells demonstrated that a unique transcriptional pattern is activated in progenitors by F36VMpl dimerization. Ligand-inducible dimerization of intracellular Mpl in human myelo-erythroid progenitors induces progenitor expansion and erythropoiesis through molecular mechanisms that are not shared by Tpo stimulation of endogenous Mpl.

Neural stem cells (NSC) persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ stem and progenitor cell proliferation are not fully elucidated. Vitamin K-dependent proteins (VKDP) are mainly secreted factors that were initially discovered as major regulators of blood coagulation. Warfarin, a widespread anticoagulant, is a vitamin K antagonist that inhibits the production of functional VKDP. We demonstrate that the suppression of functional VKDP production, in vitro, by exposure of SVZ cell cultures to warfarin or in vivo by its intracerebroventricular injection to mice, leads to a substantial increase in SVZ cell proliferation. We identify the anti-coagulant factor, protein S and its structural homolog Gas6, as the two only VKDP produced by SVZ cells and describe the expression and activation pattern of their Tyro3, Axl, Mer tyrosine kinase receptors. Both in vitro and in vivo loss of function studies consisting in either Gas6 gene invalidation or in endogenous protein S neutralization, provided evidence for an important novel regulatory role of these two VKDP in the SVZ neurogenic niche. Specifically, we show that while a loss of Gas6 leads to a reduction in the numbers of stem-like cells and in olfactory bulb neurogenesis, endogenous protein S inhibits SVZ cell proliferation. Our study opens up new perspectives for investigating further the role of vitamin K, VKDP and anticoagulants in neural stem cell biology in health and disease.

Neural stem/progenitor cells present in the subventricular zone (SVZ) are a potencial source of repairing cells after injury. Therefore, the identification of novel players that modulate neural stem cells differentiation can have a huge impact in stem cell-based therapies. Herein, we describe a unique role of histamine in inducing functional neuronal differentiation from cultured mouse SVZ stem/progenitor cells. This proneurogenic effect depends on histamine 1 receptor activation and involves epigenetic modifications and increased expression of Mash1, Dlx2 and Ngn1 genes. Biocompatible poly (lactic-co-glycolic acid) (PLGA) microparticles, engineered to release histamine in a controlled and prolonged manner, also triggered robust neuronal differentiation in vitro. Preconditioning with histamine-loaded microparticles facilitated neuronal differentiation of SVZ-GFP cells grafted in hippocampal slices and in in vivo rodent brain. We propose that neuronal commitment triggered by histamine per se or released from biomaterial-derived vehicles may represent a new tool for brain repair strategies.

Bone resorption by osteoclasts and bone formation by osteoblasts are tightly coupled processes implicating factors in TNF, bone morphogenetic protein and Wnt families. In osteoimmunology, macrophages were described as another critical cell population regulating bone formation by osteoblasts but the coupling factors were not identified. Using a high throughput approach, we identified here Oncostatin M (OSM), a cytokine of the IL-6 family, as a major coupling factor produced by activated circulating CD14⁺ or bone marrow CD11b⁺ monocytes/macrophages that induces osteoblast differentiation and matrix mineralization from human mesenchymal stem cells (MSC) while inhibiting adipogenesis. Upon toll-like receptors (TLRs) activation by lipopolysaccharide or endogenous ligands, OSM was produced in classically activated inflammatory M1 and not M2 macrophages, through a cyclooxygenase-2 and prostaglandin-E2 regulatory loop. Stimulation of osteogenesis by activated monocytes/macrophages was prevented using neutralizing antibodies or siRNA to OSM, OSM receptor subunits gp130 and OSMR or to the downstream transcription factor STAT3. The induced osteoblast differentiation program culminated with enhanced expression of C/EBPδ (CCAAT-enhancer-binding protein δ), Cbfa1 and alkaline phosphatase. Overexpression of OSM in the tibia of mice has led to new bone apposition with no sign of bone resorption. Two other cytokines had also a potent role in bone formation induced by monocytes/macrophages and TLRs activation: IL-6 and Leukemia inhibitory factor. We propose that during bone inflammation, infection or injury, the IL-6 family signaling network activated by macrophages and TLR ligands stimulates bone formation that is largely uncoupled from bone resorption and is thus an important target for anabolic bone therapies.

Recent successes in the stem cell field have identified some of the key chemical and biological cues which drive photoreceptor derivation from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), however the efficiency of this process is variable. We have designed a three-step photoreceptor differentiation protocol combining previously published methods that direct the differentiation of hESC and hiPSC towards a retinal lineage which we further modified with additional supplements selected on the basis of reports from eye field and retinal development. We report that hESC and hiPSC differentiating under our regime over a 60 day period sequentially acquire markers associated with neural, retinal field, retinal pigmented epithelium and photoreceptor cells, including mature photoreceptor markers OPN1SW and RHODOPSIN with a higher efficiency than previously reported. In addition, we report the ability of hESC and hiPSC cultures to generate neural and retinal phenotypes under minimal culture conditions which may be linked to their ability to endogenously up-regulate the expression of a range of factors important for retinal cell type specification. However, cultures that were differentiated with full supplementation under our photoreceptor-induction regime achieve this within a significantly shorter time frame and show a substantial increase in the expression of photoreceptor-specific markers in comparison to cultures differentiated under minimal conditions. Interestingly, cultures supplemented only with B27 and/or N2 displayed comparable differentiation efficiency to those under full supplementation, indicating a key role for B27 and N2 during the differentiation process. Furthermore, our data highlight an important role for Dkk1 and Noggin in enhancing the differentiation of hESC and hiPSC towards retinal progenitor cells and photoreceptor precursors during the early stages of differentiation, while suggesting that further maturation of these cells into photoreceptors may not require additional factors and can ensue under minimal culture conditions.

OCT4 is a key transcription factor in maintaining the pluripotency and self-renewal of embryonic stem (ES) cells. Human OCT4 gene can generate three mRNA isoforms (OCT4A, OCT4B and OCT4B1) by alternative splicing and four protein isoforms (OCT4A, OCT4B-265, OCT4B-190 and OCT4B-164) by alternative splicing or alternative translation initiation. OCT4A is a transcription factor responsible for the stemness of ES cells, while the function of OCT4B protein isoforms is still not clear. We have previously reported that OCT4B-190 functioned in cell stress response. Here we present another product of OCT4 gene, OCT4B-265, is up-regulated under genotoxic stress in stem cells, and it may function in stress response through p53 signaling pathway. This work gives an insight into the novel function of OCT4B protein isoforms and helps us to understand the complex expression patterns and biological functions of OCT4 gene.

Embryonic stem cell (ES-cell) specific microRNAs (miRNAs) play a critical role in the maintenance of pluripotency and self-renewal but the complete network between these miRNAs and their broad range of target genes still remains elusive. Here we demonstrate that miR-290 cluster, the most abundant miRNA family in ES-cells, targets the NF-κB subunit p65 (also known as RelA) by repressing its translation. Forced expression of p65 causes loss of pluripotency, promotes ES-cell differentiation and leads to an epithelial to mesenchymal Transition (EMT). These data define p65 as a novel target gene of miR-290 cluster and provide new insight into the function of ES-cell specific miRNAs.

TCF4, a key transcription factor of Wnt signaling system, has been recently found to be essential for maintaining stem cells. However, its signaling pathway is not well elucidated. This study was to explore the functional roles and signaling pathway of TCF4 in maintaining adult stem cell properties using human corneal epithelial stem cells as a model. With immunofluorescent staining and real-time PCR, we observed that TCF4 was exclusively expressed in the basal layer of human limbal epithelium where corneal epithelial stem cells reside. TCF4 was found to be well co-localized with ABCG2 and p63, two recognized epithelial stem/progenitor cell markers. Using in vitro cultures models of primary human corneal epithelial cells (HCECs), we revealed that TCF4 mRNA and protein were upregulated by cells in exponential growth stage, and RNA interference by siRNA-TCF4 (10-50nM) transfection blocked TCF4 signaling and suppressed cell proliferation as measured by WST-1 assay. TCF4 silence was found to be accompanied by downregulated proliferation associated factors p63 and survivin, as well as upregulated cyclin-dependent kinase inhibitor 1C (p57). By creating a wound healing model in vitro, we identified upregulation and activation of β-catenin/TCF4 with their protein translocation from cytoplasm to nuclei, as evaluated by RT-qPCR, immunostaining and Western blotting. Upregulated p63/survivin and downregulated p57 were further identified to be TCF4 downstream molecules that promote cell migration and proliferation in wound healing process. These findings demonstrate that transcription factor TCF4 plays an important role in determining or maintaining the phenotype and functional properties of human corneal epithelial stem cells.

The Notch pathway plays a pivotal role in regulating cell fate decisions in many stem cell systems. However, the full repertoire of Notch target genes in vivo and the mechanisms through which this pathway activity is integrated with other signaling pathways are largely unknown. Here, we report a transgenic mouse in which the activation of the Notch pathway massively expands the neural stem cell (NSC) pool in a cell context-dependent manner. Using this in vivo system, we identify direct targets of RBPJ/N1ICD in cortical NSCs at a genome-wide level through combined ChIP-Seq and transcriptome analyses. Through a highly conservative analysis of these data sets, we identified 98 genes that are directly regulated by N1ICD/RPBJ in vivo. These include many transcription factors that are known to be critical for NSC self-renewal (Sox2, Pax6, Tlx, and Id4) and the transcriptional effectors of the Wnt, SHH and Hippo pathways, TCF4, Gli2, Gli3, Yap1, and Tead2. Since little is known about the function of the Hippo-Yap pathway in NSCs, we analyzed Yap1 expression and function in NSCs. We show that Yap1 expression is restricted to the stem cell compartment in the developing forebrain and that its expression is sufficient to rescue Notch pathway inhibition in NSC self-renewal assays. Together, the results of this study reveal a previously underappreciated complexity and breadth of Notch1 targets in vivo and show direct interaction between Notch and Hippo-Yap pathways in NSCs.

Maintaining adequate numbers of spermatogonial stem cells is required for the production of the millions of sperm required for male fertility. To date, however, the mechanisms that regulate the size of this pool in the adult are poorly defined. Glial cell line-derived neurotrophic factor (GDNF) is required for establishing this pool in the prepubertal animal, but its in vivo function in the normal adult testis has never been examined directly. We used a chemical-genetic approach to address this issue. We generated mice carrying a single amino acid mutation (V805A) in Ret, the kinase subunit of the GDNF receptor. This mutation does not affect normal GDNF signaling, but renders it susceptible to inhibition by the ATP competitive inhibitor, NA-PP1. When GDNF signaling was blocked in adults for 11 days, only a few cells remained that expressed the stem spermatogonial markers, Gfrα1 and Zbtb16 and testicular Ret mRNA content was reduced markedly. These decreases were associated with depletion of functional stem spermatogonia; some were lost when GDNF signaling was inhibited for only 2 days while others survived for up to 11 days. However, when signaling was restored, the remaining stem cells proliferated, initiating tissue restoration. In conclusion, these results provide the first direct proof that GDNF acutely regulates the numbers of spermatogonial stem cells in the normal adult testis. Additionally, these results demonstrate different sensitivities among subpopulations of these stem cells to inhibition of GDNF signaling.

MicroRNAs (miRNAs) are short non-coding RNAs, which post-transcriptionally regulate gene expression. miRNAs are transcribed as precursors and matured to active forms by a series of enzymes, including Dicer. miRNAs are important in governing cell differentiation, development and disease. We have recently developed a feeder- and serum-free protocol for direct derivation of ECs from human embryonic stem cells (hESCs) and provided evidence of increases in angiogenesis-associated microRNAs (miR-126 and -210) during the process. However, the functional role of miRNAs in hESC differentiation to vascular endothelial cell (EC) remains to be fully interrogated.

Here, we show that the reduction of miRNA maturation induced by Dicer knockdown suppressed hES-EC differentiation. A miRNA microarray was performed to quantify hES-EC miRNA profiles during defined stages of endothelial differentiation. miR-99b, miR-181a and miR-181b was identified as increasing in a time- and differentiation-dependent manner to peak in mature hESC-ECs and adult ECs. Augmentation of miR-99b, -181a and -181b levels by lentiviral-mediated transfer potentiated the mRNA and protein expression of EC-specific markers, Pecam1 and VE Cadherin, increased nitric oxide production and improved hES-EC induced therapeutic neovascularisation in vivo. Conversely, knockdown did not impact endothelial differentiation. Our results suggest that miR-99b, -181a and -181b comprise a component of an endothelial-miRNA signature and are capable of potentiating EC differentiation from pluripotent hESCs.

Stroke remains one of the most promising targets for cell therapy. Thorough preclinical efficacy testing of human neural stem cell (hNSC) lines in a rat model of stroke (transient middle cerebral artery occlusion) is, however, required for translation into a clinical setting. Magnetic resonance imaging (MRI) here confirmed stroke damage and allowed the targeted injection of 450,000 hNSCs (CTX0E03) into peri-infarct tissue, rather than the lesion cyst. Intraparenchymal cell implants improved sensorimotor dysfunctions (bilateral asymmetry test) and motor deficits (footfault test, rotameter). Importantly, analyses based on lesion topology (striatal versus striatal+cortical damage) revealed a more significant improvement in animals with a stroke confined to the striatum. However, no improvement in learning and memory (water maze) was evident. An intracerebroventricular injection of cells did not result in any improvement. MRI-based lesion, striatal and cortical volumes were unchanged in treated animals compared to those with stroke that received an intraparenchymal injection of suspension vehicle. Grafted cells only survived after intraparenchymal injection with a striatal+cortical topology resulting in better graft survival (16,026 cells) than in animals with smaller striatal lesions (2,374 cells). Almost 20% of cells differentiated into GFAP+ astrocytes, but <2% turned into FOX3+ neurons. These results indicate that CTX0E03 implants robustly recover behavioral dysfunction over a 3 months time frame and that this effect is specific to their site of implantation. Lesion topology is potentially an important factor in the recovery, with a stroke confined to the striatum showing a better outcome compared to a larger area of damage.

Human embryonic stem cells (hESC) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic β cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15 to 20% more DE as compared to Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17 and FOXA2 when compared to Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2 and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage.

FGF, TGF/Nodal, and Insulin/IGF signaling pathways are sufficient to maintain human ES and iPS cells in a proliferative, undifferentiated state. Here we show that only a few FGF family members (FGF2, FGF4, FGF6 and FGF9) are able to sustain strong ERK phosphorylation and NANOG expression levels in human ES cells. Surprisingly, FGF1, which is reported to target the same set of receptors as FGF2, fails to sustain ERK phosphorylation and NANOG expression under standard culture conditions. We find that the failure of FGF1 to sustain ES is due to thermal instability of the wild type protein, not receptor specificity, and that a mutated thermal stable FGF1 sustains human ES cells, and supports both differentiation and reprogramming protocols.

Colorectal cancer (CRC) is one of the most commonly diagnosed and lethal cancers worldwide. It is a multistep process that requires the accumulation of genetic/epigenetic aberrations. There are several issues concerning colorectal carcinogenesis that remain unanswered, such as the cell of origin and the type of cells that propagate the tumor after its initiation. There are two models of carcinogenesis: the stochastic and the cancer stem cell (CSC) model. According to the stochastic model, any kind of cell is capable of initiating and promoting cancer development, whereas the CSC model suggests that tumors are hierarchically organized and only CSCs possess cancer-promoting potential. Moreover, various molecular pathways, such as Wingless/Int (Wnt) and Notch, as well as the complex crosstalk network between microenvironment and CSCs, are involved in CRC. Identification of CSCs remains controversial due to the lack of widely accepted specific molecular markers. CSCs are responsible for tumor relapse, because conventional drugs fail to eliminate the CSC reservoir. Therefore, the design of CSC-targeted interventions is a rational target, which will enhance responsiveness to traditional therapeutic strategies and reduce local recurrence and metastasis. This review discusses the implications of the newly introduced CSC model in CRC, the markers used up to now for CSC identification, and its potential implications in the design of novel therapeutic approaches. STEM CELLS2012;30:363–371

There is increasing evidence that a variety of cancers arise from transformation of normal stem cells to cancer stem cells (CSCs). CSCs are thought to sustain cancer progression, invasion, metastasis, and recurrence after therapy. Reports suggest that CSCs are highly resistant to conventional therapy. Emerging evidences show that the chemoresistance of CSCs are in part due to the activation of B cell-specific Moloney murine leukemia virus integration site 1 (BMI1), a stem cell factor, and a polycomb group family member. BMI1 is reported to regulate the proliferation activity of normal, stem, and progenitor cells. BMI1 plays a role in cell cycle, cell immortalization, and senescence. Numerous studies demonstrate that BMI1, which is upregulated in a variety of cancers, has a positive correlation with clinical grade/stage and poor prognosis. Although evidences are in support of the role of BMI1 as a factor in chemoresistance displayed by CSCs, its mechanism of action is not fully understood. In this review, we provide summary of evidences (with mechanism of action established) suggesting the significance of BMI1 in chemoresistance and recurrence of CSCs. STEM CELLS2012;30:372–378

Pluripotent stem cells can be differentiated into hematopoietic lineages in vitro and hold promise for the future treatment of hematological disease. Differentiation strategies involving defined factors in serum-free conditions have been successful in producing hematopoietic progenitors and some mature cell types from mouse and human embryonic stem cells and induced pluripotent cells. However, these precisely defined protocols are relatively inefficient and have not been used successfully to produce hematopoietic stem cells capable of multilineage long-term reconstitution of the hematopoietic system. More complex differentiation induction strategies including coculture with stromal cells derived from sites of hematopoietic activity in vivo and enforced expression of reprogramming transcription factors, such as HOXB4, have been required to increase the efficiency of the differentiation procedure and to produce these most potent hematopoietic stem cells. We review the studies that have used HOXB4 to improve hematopoietic differentiation from pluripotent cells focusing on studies that have provided some insight into its mechanism of action. A better understanding of the molecular pathways involved in the action of HOXB4 might lead to more defined culture systems and safer protocols for clinical translation. STEM CELLS2012;30:379–385

The vasculature, an organ that penetrates every other organ, is ideally poised to be the site where pools of stem cells are placed, to be deployed and committed in response to feedback regulation, and to respond to demands for new vascular structures. These pools of multipotent cells are often under the regulation of various members of the transforming growth factor-β superfamily, including the bone morphogenetic proteins and their antagonists. Regulation of stem cell populations affects their recruitment, differentiation, spatial organization, and their coordination with host tissue. Loss and dysregulation of feedback control cause a variety of diseases that involve ectopic tissue formation, including atherosclerotic lesion formation and calcification, diabetic vasculopathies, and arteriovenous malformations. STEM CELLS2012;30:386–391

Recent studies have identified gene signatures in malignant tumors that are associated with human embryonic stem cells, suggesting a molecular relationship between aggressive cancers and pluripotency. Here, we characterize neural precursors (NPs) derived from transformed human embryonic stem cells (N-t-hESCs) that exhibit neoplastic features of human brain tumors. NPs derived from t-hESCs have enhanced cell proliferation and an inability to mature toward the astrocytic lineage, compared with progeny derived from normal human embryonic stem cells (N-hESCs) independent of adherent or neurosphere outgrowth. Intracranial transplantation of NPs derived from N-t-hESCs and N-hESCs into NOD SCID mice revealed development of neuroectoderm tumors exclusively from the N-t-hESCs NPs and not from normal N-hESCs. These tumors infiltrated the ventricles and the cerebellum of recipient mice and displayed morphological, phenotypic, and molecular features associated with classic medulloblastoma including retention of a pluripotent signature. Importantly, N-t-hESCs did not exhibit cytogenetic changes associated with medulloblastoma, suggesting that aberrant cellular and molecular properties precede the acquisition of karyotypic changes thus underscoring the value of this model system of human medulloblastoma. Our study demonstrates that NPs from a starting population of neoplastic human pluripotent parent cells possess brain tumor-initiating cell capacity, thereby providing a model system to investigate initiation and progression of primitive human neural cancers that are difficult to assess using somatic sources. STEM CELLS2012;30:392–404

Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. Although many GBM tumors are believed to be caused by self-renewing, glioblastoma-derived stem-like cells (GSCs), the mechanisms that regulate self-renewal and other oncogenic properties of GSCs are only now being unraveled. Here we showed that GSCs derived from GBM patient specimens express varying levels of the transcriptional repressor repressor element 1 silencing transcription factor (REST), suggesting heterogeneity across different GSC lines. Loss- and gain-of-function experiments indicated that REST maintains self-renewal of GSCs. High REST-expressing GSCs (HR-GSCs) produced tumors histopathologically distinct from those generated by low REST-expressing GSCs (LR-GSCs) in orthotopic mouse brain tumor models. Knockdown of REST in HR-GSCs resulted in increased survival in GSC-transplanted mice and produced tumors with higher apoptotic and lower invasive properties. Conversely, forced expression of exogenous REST in LR-GSCs produced decreased survival in mice and produced tumors with lower apoptotic and higher invasive properties, similar to HR-GSCs. Thus, based on our results, we propose that a novel function of REST is to maintain self-renewal and other oncogenic properties of GSCs and that REST can play a major role in mediating tumorigenicity in GBM. STEM CELLS2012;30:405–414

Resistance to anticancer therapy has been attributed to interindividual differences in gene expression pathways among tumors, and to the existence within tumors of cancer stem cells with self-renewal capacity. In previous studies, we have demonstrated that the human embryonic stem cell (hESC)-derived cellular microenvironment in immunocompromised mice enables functional distinction of heterogeneous tumor cells, including cells that do not grow into a tumor in conventional direct tumor xenograft platform. In the current study, we use clonally expanded subpopulations derived from ovarian clear cell carcinoma of a single tumor, to demonstrate striking intratumoral phenotypic heterogeneity that is dynamically dependent on the tumor growth microenvironment. Each of six clonally expanded subpopulations displays a different level of morphologic and tumorigenic differentiation, wherein growth in the hESC-derived microenvironment favors growth of CD44+ aldehyde dehydrogenase positive pockets of self-renewing cells that sustain tumor growth through a process of tumorigenic differentiation into CD44− aldehyde dehydrogenase negative derivatives. Strikingly, these derivative cells display microenvironment-dependent plasticity with the capacity to restore self-renewal and CD44 expression. Such intratumoral heterogeneity and plasticity at the level of the key properties of self-renewal and tumorigenic differentiation suggests that a paradigm shift is needed in the approach to anticancer therapy, with the aim of turning malignant growth into a chronic manageable disorder, based on continual monitoring of these tumor growth properties. The hESC-based in vivo model renders intratumoral heterogeneity in the self-renewal and tumorigenic differentiation amenable to biological analysis as well as anticancer therapy testing. STEM CELLS2012;30:415–424

Neural differentiation of embryonic stem cells (ESCs) requires coordinated repression of the pluripotency regulatory program and reciprocal activation of the neurogenic regulatory program. Upon neural induction, ESCs rapidly repress expression of pluripotency genes followed by staged activation of neural progenitor and differentiated neuronal and glial genes. The transcriptional factors that underlie maintenance of pluripotency are partially characterized whereas those underlying neural induction are much less explored, and the factors that coordinate these two developmental programs are completely unknown. One transcription factor, REST (repressor element 1 silencing transcription factor), has been linked with terminal differentiation of neural progenitors and more recently, and controversially, with control of pluripotency. Here, we show that in the absence of REST, coordination of pluripotency and neural induction is lost and there is a resultant delay in repression of pluripotency genes and a precocious activation of both neural progenitor and differentiated neuronal and glial genes. Furthermore, we show that REST is not required for production of radial glia-like progenitors but is required for their subsequent maintenance and differentiation into neurons, oligodendrocytes, and astrocytes. We propose that REST acts as a regulatory hub that coordinates timely repression of pluripotency with neural induction and neural differentiation. STEM CELLS2012;30:425–434

Mutations in human induced pluripotent stem cells (iPSCs) pose a risk for their clinical use due to preferential reprogramming of mutated founder cell and selection of mutations during maintenance of iPSCs in cell culture. It is unknown, however, if mutations in iPSCs are due to stress associated with oncogene expression during reprogramming. We performed whole exome sequencing of human foreskin fibroblasts and their derived iPSCs at two different passages. We found that in vitro passaging contributed 7% to the iPSC coding point mutation load, and ultradeep amplicon sequencing revealed that 19% of the mutations preexist as rare mutations in the parental fibroblasts suggesting that the remaining 74% of the mutations were acquired during cellular reprogramming. Simulation suggests that the mutation intensity during reprogramming is ninefold higher than the background mutation rate in culture. Thus the factor induced reprogramming stress contributes to a significant proportion of the mutation load of iPSCs. STEM CELLS2012;30:435–440

Our understanding of human germ cell development is limited in large part due to inaccessibility of early human development to molecular genetic analysis. Pluripotent human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been shown to differentiate to cells of all three embryonic germ layers, as well as germ cells in vitro, and thus may provide a model for the study of the genetics and epigenetics of human germline. Here, we examined whether intrinsic germ cell translational, rather than transcriptional, factors might drive germline formation and/or differentiation from human pluripotent stem cells in vitro. We observed that, with overexpression of VASA (DDX4) and/or DAZL (Deleted in Azoospermia Like), both hESCs and iPSCs differentiated to primordial germ cells, and maturation and progression through meiosis was enhanced. These results demonstrate that evolutionarily unrelated and divergent RNA-binding proteins can promote meiotic progression of human-derived germ cells in vitro. These studies describe an in vitro model for exploring specifics of human meiosis, a process that is remarkably susceptible to errors that lead to different infertility-related diseases. STEM CELLS2012;30:441–451

Human embryonic stem cells (hESC) have a unique capacity to self-renew and differentiate into all the cell types found in human body. Although the transcriptional regulators of pluripotency are well studied, the role of cytoplasmic regulators is still poorly characterized. Here, we report a new stem cell-specific RNA-binding protein L1TD1 (ECAT11, FLJ10884) required for hESC self-renewal and cancer cell proliferation. Depletion of L1TD1 results in immediate downregulation of OCT4 and NANOG. Furthermore, we demonstrate that OCT4, SOX2, and NANOG all bind to the promoter of L1TD1. Moreover, L1TD1 is highly expressed in seminomas, and depletion of L1TD1 in these cancer cells influences self-renewal and proliferation. We show that L1TD1 colocalizes and interacts with LIN28 via RNA and directly with RNA helicase A (RHA). LIN28 has been reported to regulate translation of OCT4 in complex with RHA. Thus, we hypothesize that L1TD1 is part of the L1TD1-RHA-LIN28 complex that could influence levels of OCT4. Our results strongly suggest that L1TD1 has an important role in the regulation of stemness. STEM CELLS2012;30:452–460

Unlike mouse embryonic stem cells (ESCs), which are closely related to the inner cell mass, human ESCs appear to be more closely related to the later primitive ectoderm. For example, human ESCs and primitive ectoderm share a common epithelial morphology, growth factor requirements, and the potential to differentiate to all three embryonic germ layers. However, it has previously been shown that human ESCs can also differentiate to cells expressing markers of trophoblast, an extraembryonic lineage formed before the formation of primitive ectoderm. Here, we show that phorbol ester 12-O-tetradecanoylphorbol 13-acetate causes human ESCs to undergo an epithelial mesenchymal transition and to differentiate into cells expressing markers of parietal endoderm, another extraembryonic lineage. We further confirmed that this differentiation is through the activation of protein kinase C (PKC) pathway and demonstrated that a particular PKC subtype, PKC-δ, is most responsible for this transition. STEM CELLS2012;30:461–470

Mesenchymal stem cells (MSCs) are considered as an attractive source of cells for cartilage engineering due to their availability and capacity for expansion and multipotency. Differentiation of MSC into chondrocytes is crucial to successful cartilage regeneration and can be induced by various biological agents, including polysaccharides that participate in many biological processes through interactions with growth factors. Here, we hypothesize that growth factor-induced differentiation of MSC can be increased by chemically oversulfated marine polysaccharides. To test our hypothesis, human adipose tissue-derived MSCs (hATSCs) were cultured in pellets with transforming growth factor (TGF)-β1-supplemented chondrogenic medium containing either the polysaccharide GY785 DR or its oversulfated isoform GY785 DRS. Chondrogenesis was monitored by the measurement of pellet volume, quantification of DNA, collagens, glycosaminoglycans (GAGs), and immunohistological staining. Our data revealed an increase in pellet volume, total collagens, and GAG production with GY785 DRS and chondrogenic medium. The enhanced chondrogenic differentiation of hATSC was further demonstrated by the increased expression of several chondrogenic markers by real-time reverse transcription-polymerase chain reaction. In addition, surface plasmon resonance analyses revealed that TGF-β1 bound GY785 DRS with higher affinity compared to GY785 DR. In association with TGF-β1, GY785 DRS was found to upregulate the phosphorylation of extracellular signal-regulated kinase 1/2, indicating that oversulfated polysaccharide affects the mitogen activated protein kinase signaling activity. These results demonstrate the upregulation of TGF-β1-dependent stem cell chondrogenesis by a chemically oversulfated marine polysaccharide. This polysaccharide of marine origin is easily producible and therefore could be considered a promising additive to drive efficient and reliable MSC chondrogenesis for cartilage tissue engineering. STEM CELLS2012;30:471–480

Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion. STEM CELLS2012;30:481–490

Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM. STEM CELLS2012;30:491–499

The generation of new neurons in the mammalian hippocampus continues throughout life, and lineage progression is regulated by transcription factors, local cues, and environmental influences. The ability to direct stem/progenitor cell fate in situ may have therapeutic potential. Using an in vivo retroviral delivery and lineage tracing approach, we compare the lineage-instruction factors, Pax6 and Olig2, and demonstrate that both participate in regulation of adult hippocampal neurogenesis in adult rats. We show that overexpression of the proneuronal factor Pax6 pushes neuronal precursor cells to early maturation and increases the frequency of neuronal phenotypes. However, Pax6 overexpression results in no net increase in neurogenesis at 3 weeks. Blocking of Olig2 function reduces and slows neuronal commitment and differentiation and decreases net neurogenesis. Altering expression of both factors also changes gliogenesis. Our results establish that Pax6 decreases the number of Neuron-Glia 2 progenitor cells and prevents oligodendrocytic lineage commitment, while repression of Olig2 results in an expanded astrocytic lineage. We conclude that selectively modifying transcriptional cues within hippocampal progenitor cells is sufficient to induce a cell fate switch, thus altering the neurogenesis–gliogenesis ratio. In addition, our data show the competence of multiple progenitor lineages to respond divergently to the same signal. Therefore, directing instructive cues to select phenotype and developmental stage could be critical to achieve precise outcomes in cell genesis. Further understanding the regulation of lineage progression in all progenitor populations within the target region will be important for developing therapeutic strategies to direct cell fate for brain repair. STEM CELLS2012;30:500–509

Inflammatory mediators, many of which activate the signaling of nuclear factor kappa B (NFκB), have received increasing attention in the field of neurogenesis. NFκB signaling regulates neurite outgrowth and neural plasticity as well as the proliferation/apoptosis and terminal differentiation of neural stem cells (NSCs). Early neurogenesis from NSCs produces identical progeny through symmetric division and committed daughter cells through asymmetric division. Here, we show that NFκB signaling is required for NSC initial differentiation. The canonical IKKβ/IκBα/p65 pathway is activated during the initial stages of neural differentiation induced by treatment with TNFα or withdrawal of epidermal growth factor/basic fibroblast growth factor. NSC-specific inhibition of NFκB in transgenic mice causes an accumulation of Nestin⁺/Sox2⁺/glial fibrillary acidic protein⁺ NSCs. Inhibition of NFκB signaling in vitro blocks differentiation and asymmetric division and maintains NSCs in an undifferentiated state. The induction of initial differentiation and asymmetry by NFκB signaling occurs through the inhibition of C/EBPβ expression. Our data reveal a novel function of NFκB signaling in early neurogenesis and provide insight into the molecular mechanisms underlying neurodevelopmental disorders and neurodegenerative diseases. STEM CELLS2012;30:510–524

Local renin–angiotensin systems (RASs) regulate the differentiation of tissue progenitors. However, it is not known whether such systems can regulate the development of pancreatic progenitor cells (PPCs). To address this issue, we characterized the expression profile of major RAS components in human fetal PPC preparations and examined their effects on the differentiation of PPCs into functional islet-like cell clusters (ICCs). We found that expression of RAS components was highly regulated throughout PPC differentiation and that locally generated angiotensin II (Ang II) maintained PPC growth and differentiation via Ang II type 1 and type 2 (AT₁ and AT₂) receptors. In addition, we observed colocalization of AT₂ receptors with critical β-cell phenotype markers in PPCs/ICCs, as well as AT₂ receptor upregulation during differentiation, suggesting that these receptors may regulate β-cell development. In fact, we found that AT₂, but not AT₁, receptor was a key mediator of Ang II-induced upregulation of transcription factors important in β-cell development. Furthermore, lentivirus-mediated knockdown of AT₂ receptor suppressed the expression of these transcription factors in ICCs. Transplantation of AT₂ receptor-depleted ICCs into immune-privileged diabetic mice failed to ameliorate hyperglycemia, implying that AT₂ receptors are indispensable during ICC maturation in vivo. These data strongly indicate that a local RAS is involved in governing the functional maturation of pancreatic progenitors toward the endocrine lineage. STEM CELLS2012;30:525–536

The cyclin-dependent kinase inhibitor p21^waf1/cip mediates the p53-dependent G1/S checkpoint, which is generally considered to be a critical requirement to maintain genomic stability after DNA damage. We used staggered 5-ethynyl-2′deoxyuridine/5-bromo-2′-deoxyuridine double-labeling in vivo to investigate the cell cycle progression and the role of p21^waf1/cip in the DNA damage response of neural stem and progenitor cells (NSPCs) after exposure of the developing mouse cortex to ionizing radiation. We observed a radiation-induced p21-dependent apoptotic response in migrating postmitotic cortical cells. However, neural stem and progenitor cells (NSPCs) did not initiate a p21^waf1/cip1-dependent G1/S block and continued to enter S-phase at a similar rate to the non-irradiated controls. The G1/S checkpoint is not involved in the mechanisms underlying the faithful transmission of the NSPC genome and/or the elimination of critically damaged cells. These processes typically involve intra-S and G2/M checkpoints that are rapidly activated after irradiation. p21 is normally repressed in neural cells during brain development except at the G1 to G0 transition. Lack of activation of a G1/S checkpoint and apoptosis of postmitotic migrating cells after DNA damage appear to depend on the expression of p21 in neural cells, since substantial cell-to-cell variations are found in the irradiated cortex. This suggests that repression of p21 during brain development prevents the induction of the G1/S checkpoint after DNA damage. STEM CELLS2012;30:537–547

Defining the signaling mechanisms that regulate the fate of adult stem cells is an essential step toward their use in regenerative medicine. Platelet-derived growth factor receptor (PDGFR) signaling plays a crucial role in specifying mesenchymal stem cell (MSC) commitment to mesenchymal lineages. Based on the hypothesis that selective inhibition of signaling pathways involved in differentiation may increase stem cell potency, we examined the role of PDGFR signaling in controlling the fate of human MSCs. Using a small molecular PDGFR inhibitor that induced MSCs toward a more rounded shape, expression of Oct4 and Nanog were markedly upregulated. In these PDGFR inhibitor-treated MSCs, Oct4 and Nanog expression and cell shape were regulated by janus kinase (JAK), MAPK kinase (MEK), and epidermal growth factor receptor (EGFR) signaling. Under defined differentiation conditions, these PDGFR-inhibited MSCs expressed definitive endodermal, ectodermal, and mesodermal markers. We also confirmed that depletion of individual PDGF receptors upregulated expression of Oct4A and Nanog. This study identifies PDGFR signaling as a key regulator of Oct4 and Nanog expression and of MSC potency. Thus, inhibiting these specific receptor tyrosine kinases, which play essential roles in tissue formation, offers a novel approach to unlock the therapeutic capacity of MSCs. STEM CELLS2012;30:548–560

Oxygen levels in tissues including the embryonic brain are lower than those in the atmosphere. We reported previously that Notch signal activation induces demethylation of astrocytic genes, conferring astrocyte differentiation ability on midgestational neural precursor cells (mgNPCs). Here, we show that the oxygen sensor hypoxia-inducible factor 1α (HIF1α) plays a critical role in astrocytic gene demethylation in mgNPCs by cooperating with the Notch signaling pathway. Expression of constitutively active HIF1α and a hyperoxic environment, respectively, promoted and impeded astrocyte differentiation in the developing brain. Our findings suggest that hypoxia contributes to the appropriate scheduling of mgNPC fate determination. STEM CELLS2012;30:561–569

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by retroviral overexpression of the transcription factors Oct4, Sox2, Klf4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk for chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC, albeit at lower efficiency. To elucidate the influence of factor reduction on subsequent differentiation, we compared the efficiency of neuronal differentiation in iPSC generated from postnatal murine neural stem cells with either one (Oct4; iPSC_1F-NSC), two (Oct4, Klf4; iPSC_2F-NSC), or all four factors (iPSC_4F-NSC) with those of embryonic stem cells (ESCs) and iPSC produced from fibroblasts with all four factors (iPSC_4F-MEF). After 2 weeks of coculture with PA6 stromal cells, neuronal differentiation of iPSC_1F-NSC and iPSC_2F-NSC was less efficient compared with iPSC_4F-NSC and ESC, yielding lower proportions of colonies that stained positive for early and late neuronal markers. Electrophysiological analyses after 4 weeks of differentiation identified functional maturity in neurons differentiated from ESC, iPSC_2F-NSC, iPSC_4F-NSC, and iPSC_4F-MEF but not in those from iPSC_1F-NSC. Similar results were obtained after hematoendothelial differentiation on OP9 bone marrow stromal cells, where factor-reduced iPSC generated lower proportions of colonies with hematoendothelial progenitors than colonies of ESC, iPSC_4F-NSC, and iPSC_4F-MEF. We conclude that a reduction of reprogramming factors does not only reduce reprogramming efficiency but may also worsen subsequent differentiation and hinder future application of iPSC in cell replacement therapies. STEM CELLS2012;30:570–579

'In this report, we use single cell gene expression to identify transcriptional patterns emerging during the differentiation of human embryonic stem cells (hESCs) into the endodermal lineage. Endoderm-specific transcripts are highly variable between individual CXCR4⁺ endodermal cells, suggesting that either the cells generated from in vitro differentiation are distinct or that these embryonic cells tolerate a high degree of transcript variability. Housekeeping transcripts, on the other hand, are far more consistently expressed within the same cellular population. However, when we compare the levels of housekeeping transcripts between hESCs and derived endoderm, patterns emerge that can be used to clearly separate the two embryonic cell types. We further compared four additional human cell types, including 293T, induced pluripotent stem cell (iPSC), HepG2, and endoderm-derived iPSC. In each case, the relative levels of housekeeping transcripts defined a particular cell fate. Interestingly, we find that three transcripts, LDHA, NONO, and ACTB, contribute the most to this diversity and together serve to segregate all six cell types. Overall, this suggests that levels of housekeeping transcripts, which are expressed within all cells, can be leveraged to distinguish between human cell types and thus may serve as important biomarkers for stem cell biology and other disciplines. STEM CELLS2012;30:580–584