The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming

Metabolism is vital to every aspect of cell function, yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report, using an untargeted metabolomics approach, that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells, and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis, and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly, the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs, which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs, and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency, and in evaluating iPSC and ESC equivalence.     

Regulation of embryonic and induced pluripotency by aurora kinase-p53 signaling

Many signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A?(Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity?that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming.     

Fatty acid synthesis is critical for stem cell pluripotency via promoting mitochondrial fission

Pluripotent stem cells are known to display distinct metabolic phenotypes than their somatic counterparts. While accumulating studies are focused on the roles of glucose and amino acid metabolism in facilitating pluripotency, little is known regarding the role of lipid metabolism in regulation of stem cell activities. Here, we show that fatty acid (FA) synthesis activation is critical for stem cell pluripotency. Our initial observations demonstrated enhanced lipogenesis in pluripotent cells and during cellular reprogramming. Further analysis indicated that de novo FA synthesis controls cellular reprogramming and embryonic stem cell pluripotency through mitochondrial fission. Mechanistically, we found that de novo FA synthesis regulated by the lipogenic enzyme ACC1 leads to the enhanced mitochondrial fission via (i) consumption of AcCoA which affects acetylation‐mediated FIS1 ubiquitin–proteasome degradation and (ii) generation of lipid products that drive the mitochondrial dynamic equilibrium toward fission. Moreover, we demonstrated that the effect of Acc1 on cellular reprogramming via mitochondrial fission also exists in human iPSC induction. In summary, our study reveals a critical involvement of the FA synthesis pathway in promoting ESC pluripotency and iPSC formation via regulating mitochondrial fission.     

Induced pluripotent stem cells derived from human amnion in chemically defined conditions

Fetal stem cells are a unique type of adult stem cells that have been suggested to be broadly multipotent with some features of pluripotency. Their clinical potential has been documented but their upgrade to full pluripotency could open up a wide range of cell-based therapies particularly suited for pediatric tissue engineering, longitudinal studies or disease modeling. Here we describe episomal reprogramming of mesenchymal stem cells from the human amnion to pluripotency (AM-iPSC) in chemically defined conditions. The AM-iPSC expressed markers of embryonic stem cells, readily formed teratomas with tissues of all three germ layers present and had a normal karyotype after around 40 passages in culture. We employed novel computational methods to determine the degree of pluripotency from microarray and RNA sequencing data in these novel lines alongside an iPSC and ESC control and found that all lines were deemed pluripotent, however, with variable scores. Differential expression analysis then identified several groups of genes that potentially regulate this variability in lines within the boundaries of pluripotency, including metallothionein proteins. By further studying this variability, characteristics relevant to cell-based therapies, like differentiation propensity, could be uncovered and predicted in the pluripotent stage.     

Porcine induced pluripotent stem cells analogous to naïve and primed embryonic stem cells of the mouse

Authentic or na?ve embryonic stem cells (ESC) have probably never been derived from the inner cell mass (ICM) of pig blastocysts, despite over 25 years of effort. Recently, several groups, including ours, have reported induced pluripotent stem cells (iPSC) from swine by reprogramming somatic cells with a combination of four factors, OCT4 (POU5F1)/SOX2/KLF4/c-MYC delivered by retroviral transduction. The porcine (p) iPSC resembled human (h) ESC and the mouse "Epiblast stem cells" (EpiSC) in their colony morphology and expression of pluripotent genes, and are likely dependent on FGF2/ACTIVIN/NODAL signaling, therefore representing a primed ESC state. These cells are likely to advance swine as a model in biomedical research, since grafts could potentially be matched to the animal that donated the cells for re-programming. The objective of the present work has been to develop na?ve piPSC. Employing a combination of seven reprogramming factors assembled on episomal vectors, we successfully reprogrammed porcine embryonic fibroblasts on a modified LIF-medium supplemented with two kinase inhibitors; CHIR99021, which inhibits GSK-3beta, and PD0325901, a MEK inhibitor. The derived piPSC bear a striking resemblance to na?ve mESC in colony morphology, are dependent on LIF to maintain an undifferentiated phenotype, and express markers consistent with pluripotency. They exhibit high telomerase activity, a short cell cycle interval, and a normal karyotype, and are able to generate teratomas. Currently, the competence of these lines for contributing to germ-line chimeras is being tested.     

The aging signature: a hallmark of induced pluripotent stem cells?

The discovery that somatic cells can be induced into a pluripotent state by the expression of reprogramming factors has enormous potential for therapeutics and human disease modeling. With regard to aging and rejuvenation, the reprogramming process resets an aged, somatic cell to a more youthful state, elongating telomeres, rearranging the mitochondrial network, reducing oxidative stress, restoring pluripotency, and making numerous other alterations. The extent to which induced pluripotent stem cell (iPSC)s mime embryonic stem cells is controversial, however, as iPSCs have been shown to harbor an epigenetic memory characteristic of their tissue of origin which may impact their differentiation potential. Furthermore, there are contentious data regarding the extent to which telomeres are elongated, telomerase activity is reconstituted, and mitochondria are reorganized in iPSCs. Although several groups have reported that reprogramming efficiency declines with age and is inhibited by genes upregulated with age, others have successfully generated iPSCs from senescent and centenarian cells. Mixed findings have also been published regarding whether somatic cells generated from iPSCs are subject to premature senescence. Defects such as these would hinder the clinical application of iPSCs, and as such, more comprehensive testing of iPSCs and their potential aging signature should be conducted.     

An Experimental Approach to the Generation of Human Embryonic Stem Cells Equivalents

Recently, particular attention has been paid to the human embryonic stem cells (hESC) in the context of their potential application in regenerative medicine; however, ethical concerns prevent their clinical application. Induction of pluripotency in somatic cells seems to be a good alternative for hESC recruitment regarding its potential use in tissue regeneration, disease modeling, and drug screening. Since Yamanaka’s team in 2006 restored pluripotent state of somatic cells for the first time, a significant progress has been made in the area of induced pluripotent stem cells (iPSC) generation. Here, we review the current state of knowledge in the issue of techniques applied to establish iPSC. Somatic cell nuclear transfer, cell fusion, cell extracts reprogramming, and techniques of direct reprogramming are described. Retroviral and lentiviral transduction are depicted as ways of cell reprogramming with the use of integrating vectors. Contrary to them, adenoviruses, plasmids, single multiprotein expression vectors, and PiggyBac transposition systems are examples of non-integrative vectors used in iPSC generation protocols. Furthermore, reprogramming with the delivery of specific proteins, miRNA or small chemical compounds are presented. Finally, the changes occurring during the reprogramming process are described. It is concluded that subject to some limitations iPSC could become equivalents for hESC in regenerative medicine.     

Mechanical loading prevents the stimulating effect of IL-1β on osteocyte-modulated osteoclastogenesis

Ever since the technique of coaxing ordinary skin cells into becoming pluripotent stem cells (iPSCs) has been developed, which have the potential to become any cell or tissue in the body, efforts were made to improve the approach because some major challenges. Increasing evidence suggests that several microRNAs (miRNAs) are involved in early embryonic development and embryonic stem cell formation, known as embryonic stem cell (ESC)-specific miRNAs, particularly the miR-302 family. We summarized here a novel approach to generate iPSCs by using miR-302 and its related miRNAs such as miR-367. The development of this miR-302/367-mediated iPSC (termed mirPSC) may provide tools to deal with the obstacles facing some current iPSC reprogramming methods. The mechanism by which miR-302/367 induce iPSC reprogramming is proposed.     

Somatic cell transformation into stem cell-like cells induced by different microenvironments

Development of induced pluripotent stem cell (iPSC) technology introduced a novel way to derive pluripotent stem cells, but the genetic manipulation required to generate iPSCs may lead to uncontrolled tumorigenesis of the established cells and thus limit clinical feasibility of the technology. Numerous attempts have been made to date, and alternative reprogramming of somatic cells to reactivate cellular plasticity after differentiation has been suggested. As a result, it had become clear that cell-to-cell interactions and specific acellular environments can be utilized for somatic cell reprogramming. In our previous studies, embryonic stem cell (ESC)-like cells could be derived from transforming ovarian cells and fetal fibroblasts by cell-to-cell interaction or specific cell-mediated microenvironmental factor(s). This cellular event was induced without undertaking genetic manipulation of progenitor cells. Several differences were found between the cellular properties of niche-induced, ESC-like cells and those of genetically manipulated iPSCs and the referenced ESCs. Thus, we provided evidence that terminally differentiated somatic cells either acquire pluripotency-like activity or possess cellular and genetic plasticity under a specific microenvironment and/or cell-to-cell interaction. In this minireview, we discuss derivation of stem cell-like cells under specific microenvironmental conditions in terms of technical perspectives and limitations.     

诱导多能干细胞技术的优化及其应用前景

诱导多能干细胞(induced pluripotent stem cells, iPSCs)是类似胚胎干细胞的一种细胞类型,可以通过对已分化的体细胞进行诱导重编程获得,具有自我更新能力和多潜能性,在体外疾病模型的建立、移植替代治疗、发育学等方面有广阔的应用前景,但致瘤性、转化率低、疾病模型拟合度差等缺点限制着iPS技术在临床和科研上的推广。对近几年诱导多能干细胞技术优化方面取得的新进展进行综述,重点阐述降低致瘤性和提高转化率的几种方法及iPS在临床和科研上的应用前景。