Somatic Cell Plasticity and Niemann-Pick Type C2 Protein: ADIPOCYTE DIFFERENTIATION AND FUNCTION*

The phenotypic stability of somatic cells is essential for the maintenance of both structural and functional organ integrity of the adult human body. Deregulated cell plasticity could result in the development of debilitating diseases such as cancer, fibrosis, atherosclerosis, obesity, and type 2 diabetes. We have previously demonstrated that a nonsense mutation in the NPC2 gene, which encodes ubiquitous, highly conserved, secretory protein with unknown function, leads to activation of human skin fibroblasts. The activated fibroblasts, also known as myofibroblasts, have the properties of mesenchymal stem cells and are able to differentiate along the mesodermal and endodermal lineages. Here we show that NPC2-null, but not the normal skin fibroblasts, possess characteristics of adipogenic progenitors as demonstrated by their specific gene expression pattern as well as the ability for efficient differentiation into white adipocytes. The presence of NPC2 in mature white adipocytes was also necessary for their maintenance because silencing NPC2 in differentiated cells by siRNA stimulated PPARG expression, which was followed by a shift toward a more favorable, brown adipocyte-like metabolic state characterized by up-regulated lipolysis and increased insulin sensitivity. It appears that NPC2 controls both the adipogenesis and the metabolic state of mature white adipocytes through a common mechanism that is linked to activation of FGFR2 that could be followed by induction of PPARG expression. Altogether, the current study highlights NPC2 as a novel intracrine/autocrine factor that controls adipocyte differentiation and function as well as potential therapeutic target for the treatment of type 2 diabetes and related metabolic disorders.     

Recruitment of brown fat and conversion of white into brown adipocytes: Strategies to fight the metabolic complications of obesity?

The role of white and brown adipose tissues in energy metabolism is well established. However, the existence of brown fat in adult humans was until very recently a matter of debate, and the molecular mechanisms underlying brown adipocyte development remained largely unknown. In 2009, several studies brought direct evidence for functional brown adipose tissue in adults. New factors involved in brown fat cell differentiation have been identified. Moreover, work on the origin of fat cells took an unexpected path with the recognition of different populations of brown fat cell precursors according to the anatomical location of the fat depots: a precursor common to skeletal muscle cells and brown adipocytes from brown fat depots, and a progenitor cell common to white adipocytes and brown adipocytes that appear in certain conditions in white fat depots. There is also mounting evidence that mature white adipocytes, including human fat cells, can be converted into brown fat-like adipocytes, and that the typical fatty acid storage phenotype of white adipocyte can be altered towards a fat utilization phenotype. These data open up new opportunities for the development of drugs for obesity and its metabolic and cardiovascular complications.     

Comparative analysis of human UCB and adipose tissue derived mesenchymal stem cells for their differentiation potential into brown and white adipocytes

The differentiation potential of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) into brown and white adipocytes in comparison to Adipose tissue derived MSCs (AD-MSCs) were investigated in order to characterize their potency for future cell therapies. MSCs were isolated from ten UCB samples and six liposuction materials. MSCs were differentiated into white and brown adipocytes after characterization by flow cytometry. Differentiated adipocytes were stained with Oil Red O and hematoxylin/eosin. The UCP1 protein levels in brown adipocytes were investigated by immunofluoresence and western blot analysis. Cells that expressed mesenchymal stem cells markers (CD34?, CD45?, CD90+ and CD105+) were successfully isolated from UCB and adipose tissue. Oil Red O staining demonstrated that white and brown adipocytes obtained from AD-MSCs showed 85 and 61% of red pixels, while it was 3 and 1.9%, respectively for white and brown adipocytes obtained from UCB-MSCs. Fluorescence microscopy analysis showed strong uncoupling protein 1 (UCP1) signaling in brown adipocytes, especially which were obtained from AD-MSCs. Quantification of UCP1 protein amount showed 4- and 10.64-fold increase in UCP1 contents of brown adipocytes derived from UCB-MSCs and AD-MSCs, respectively in comparison to undifferentiated MSCs (P?<?0.004). UCB-MSCs showed only a little differentiation tendency into adipocytes means it is not an appropriate stem cell type to be differentiated into these cell types. In contrast, high differentiation efficiency of AD-MSCs into brown and white adipocytes make it appropriate stem cell type to use in future regenerative medicine of soft tissue disorders or fighting with obesity and its related disorders.     

In vivo differentiation of brown adipocytes in adult mice: an electron microscopic study

The differentiation of brown adipocyte precursor cells was studied in interscapular brown adipose tissue of adult mice by electron microscopy. Different stages of cell differentiation were characterized in situ. Previous autoradiographic studies suggested that interstitial cells represent the precursor cells of fully differentiated brown adipocytes. The present observations provide morphological evidence for a progressive differentiation of interstitial stem cells into mature brown adipocytes. Four typical stages of development were identified: (1) interstitial cells, (2) protoadipocytes, (3) preadipocytes, and (4) mature brown adipocytes. Interstitial stem cells were small spindle shaped cells, situated between brown adipocytes and characterized by a high nuclear-cytoplasmic ratio, the scarcity of organelles, and the absence of lipid inclusions. Protoadipocytes resembled interstitial cells except that they contained a few tiny lipid droplets in their cytoplasm. Preadipocytes had a larger cytoplasm enclosing many mitochondria and lipid droplets; the smooth endoplasmic reticulum was well developed surrounding the lipid droplets, and was closely associated with the mitochondria. Preadipocytes had the typical structure of growing cells, developing long cytoplasmic processes between and around blood capillaries. Mature brown adipocytes represented the final stage of differentiation. Almost all their cellular volume was occupied by lipid droplets and numerous mitochondria with very dense cristae. Brown adipocytes were also characterized by a tight association with blood capillaries, as expected from metabolically active cells requiring oxygen and substrates. These observations provide direct ultrastructural evidence for a progressive differentiation of interstitial cells into brown adipocytes with a continuum of intermediate cellular types.     

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.     

A Novel Feeder-Free Culture System for Human Pluripotent Stem Cell Culture and Induced Pluripotent Stem Cell Derivation

Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC) to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC) lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines.     

Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential

When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. In the present study, we examined the multilineage differentiation potential of DFAT cells. DFAT cells obtained from adipose tissues of 18 donors exhibited a fibroblast-like morphology and sustained high proliferative activity. Flow cytometric analysis revealed that DFAT cells comprised a highly homogeneous cell population compared with that of adipose-derived stem/stromal cells (ASCs), although the cell-surface antigen profile of DFAT cells was very similar to that of ASCs. DFAT cells lost expression of mature adipocytes marker genes but retained or gained expression of mesenchymal lineage-committed marker genes such as peroxisome proliferator-activated receptor gamma (PPARgamma), RUNX2, and SOX9. In vitro differentiation analysis revealed that DFAT cells could differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate culture conditions. DFAT cells also formed osteoid matrix when implanted subcutaneously into nude mice. In addition, clonally expanded porcine DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages. These results indicate that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application for cell-based therapies.     

Genome-wide profiling of microRNAs in adipose mesenchymal stem cell differentiation and mouse models of obesity

In recent years, there has been accumulating evidence that microRNAs are key regulator molecules of gene expression. The cellular processes that are regulated by microRNAs include e.g. cell proliferation, programmed cell death and cell differentiation. Adipocyte differentiation is a highly regulated cellular process for which several important regulating factors have been discovered, but still not all are known to fully understand the underlying mechanisms. In the present study, we analyzed the expression of 597 microRNAs during the differentiation of mouse mesenchymal stem cells into terminally differentiated adipocytes by real-time RT-PCR. In total, 66 miRNAs were differentially expressed in mesenchymal stem cell-derived adipocytes compared to the undifferentiated progenitor cells. To further study the regulation of these 66 miRNAs in white adipose tissue in vivo and their dependence on PPARγ activity, mouse models of genetically or diet induced obesity as well as a mouse line expressing a dominant negative PPARγ mutant were employed.     

The application of stem cell therapy and brown adipose tissue transplantation in metabolic disorders

The discovery of brown fat in adult humans has led to increased research of the thermogenic function of this tissue in various metabolic diseases. In addition, high levels of brown fat have been correlated with lower body mass index values. Therefore, increasing brown fat mass and/or activity through methods such as the browning of white fat is considered a promising strategy to prevent and treat obesity-associated diseases. Cell-based approaches using mesenchymal stromal cells and brown adipose tissue (BAT) have been utilized to directly increase BAT mass/activity through cell and tissue implantation into animals. In addition, recent studies evaluating the transplantation of human embryonic stem cells and induced pluripotent stem (iPS) cells have shown promising results in terms of positive metabolic function. In this comprehensive review, we provide a summary of the research over the past 10 years with regard to stem cell therapy and brown fat tissue transplantation for the effective treatment of metabolic syndrome. Recent advancements in stem cell methods have allowed for the production of brown adipocytes from human iPS cells, which represent an unlimited source of cellular material with which to study adipocyte development. In addition, this process is expected to be used to further explore drug- and cell-based therapies to treat obesity-related metabolic complications.     

The role of DNA repair in the pluripotency and differentiation of human stem cells

All living cells utilize intricate DNA repair mechanisms to address numerous types of DNA lesions and to preserve genomic integrity, and pluripotent stem cells have specific needs due to their remarkable ability of self-renewal and differentiation into different functional cell types. Not surprisingly, human stem cells possess a highly efficient DNA repair network that becomes less efficient upon differentiation. Moreover, these cells also have an anaerobic metabolism, which reduces the mitochondria number and the likelihood of oxidative stress, which is highly related to genomic instability. If DNA lesions are not repaired, human stem cells easily undergo senescence, cell death or differentiation, as part of their DNA damage response, avoiding the propagation of stem cells carrying mutations and genomic alterations. Interestingly, cancer stem cells and typical stem cells share not only the differentiation potential but also their capacity to respond to DNA damage, with important implications for cancer therapy using genotoxic agents. On the other hand, the preservation of the adult stem cell pool, and the ability of cells to deal with DNA damage, is essential for normal development, reducing processes of neurodegeneration and premature aging, as one can observe on clinical phenotypes of many human genetic diseases with defects in DNA repair processes. Finally, several recent findings suggest that DNA repair also plays a fundamental role in maintaining the pluripotency and differentiation potential of embryonic stem cells, as well as that of induced pluripotent stem (iPS) cells. DNA repair processes also seem to be necessary for the reprogramming of human cells when iPS cells are produced. Thus, the understanding of how cultured pluripotent stem cells ensure the genetic stability are highly relevant for their safe therapeutic application, at the same time that cellular therapy is a hope for DNA repair deficient patients.     

Derivation of multipotent nestin+/CD271−/STRO-1− mesenchymal-like precursors from human embryonic stem cells in chemically defined conditions

The successful establishment of stem cell-based therapies requires multipotent, immunocompatible stem cells, highly efficient strategies for direct differentiation, and most importantly, optimal culture conditions for large-scale expansion of such cell populations. Other than adult tissues, human embryonic stem cells (hESCs) represent another infinitely expansible source for mesenchymal stem cell (MSC) derivation. Here, we reproducibly derived a population of Nestin⁺/CD271^?/STRO-1^? mesenchymal-like precursors from hESCs (hESC-MPs) in chemically defined conditions, without requiring any serum or serum replacement of animal origin, based on a Y-27632-assisted monolayer culture system. These cells showed slim fibroblastic morphology, and satisfied the criteria of MSCs including self-renewal, the expression of multiple MSC-specific markers and the ability to differentiate into osteoblasts, adipocytes and chondrocytes. Compared with previously reported hESC-derived MSCs, our hESC-MPs were more multipotent, and could differentiate into representative derivatives of all three embryonic germ layers including mature smooth muscle cells, cardiomyocytes, functional hepatocytes and neural cells expressing various neurotransmitter phenotypes, making them an attractive cell source for regenerative medicine.     

Functional studies of mesenchymal stem cells derived from adult human adipose tissue

Recent evidence suggests that cells with the properties of human mesenchymal stem cells (hMSCs) can be derived from adult peripheral tissues, including adipose tissue, muscle and dermis. We isolated hMSCs from the stromal-vascular portion of subcutaneous adipose tissue from seven adult subjects. These cells could be readily differentiated into cells of the chondrocyte, osteocyte and adipocyte lineage demonstrating their multipotency. We studied the functional properties of hMSCs-derived adipocytes and compared them with adipocytes differentiated from hMSCs obtained from bone marrow (BM-hMSC). The two cell types displayed similar lipolytic capacity upon stimulation with catecholamines, including a pronounced antilipolytic effect mediated through alpha2A-adrenoceptors, a typical trait in human but not rodent fat cells. Furthermore, both cell types secreted the fat cell-specific factors leptin and adiponectin in comparable amounts per time unit. The fat tissue-derived hMSCs retained their differentiation capacity up to at least fifteen passages. We conclude that hMSCs derived from adult human adipose tissue can be differentiated into fully functional adipocytes with a similar, if not identical, phenotype as that observed in cells derived from BM-hMSCs. Human adipose-tissue-derived MSCs could therefore constitute an efficient and easily obtainable renewable cellular source for studies of adipocyte biology.     

Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells

Although induced pluripotent stem (iPS) cells are indistinguishable from ES cells in their expression of pluripotent markers, their differentiation into targeted cells is often limited. Here, we examined whether the limited capacity of iPS cells to differentiate into neural lineage cells could be mitigated by improving their base-line level of pluripotency, i.e. by converting them into the so-called “naive” state. In this study, we used rabbit iPS and ES cells because of the easy availability of both cell types and their typical primed state characters. Repeated passages of the iPS cells permitted their differentiation into early neural cell types (neural stem cells, neurons, and glial astrocytes) with efficiencies similar to ES cells. However, unlike ES cells, their ability to differentiate later into neural cells (oligodendrocytes) was severely compromised. In contrast, after these iPS cells had been converted to a naive-like state, they readily differentiated into mature oligodendrocytes developing characteristic ramified branches, which could not be attained even with ES cells. These results suggest that the naive-like conversion of iPS cells might endow them with a higher differentiation capacity.     

Expression of Adipocyte Biomarkers in a Primary Cell Culture Models Reflects Preweaning Adipobiology

A cohort of genes was selected to characterize the adipogenic phenotype in primary cell cultures from three tissue sources. We compared the quantitative expression of biomarkers in culture relative to their expression in vivo because the mere presence or absence of expression is minimally informative. Although all biomarkers analyzed have biochemical functions in adipocytes, the expression of some of the biomarkers varied enormously in culture relative to their expression in the adult fat tissues in vivo, i.e. inguinal fat for white adipocytes and brite cells, interscapular brown adipose tissue for brown adipocytes, and ear mesenchymal stem cells for white adipocytes from adult mice. We propose that the pattern of expression in vitro does not reflect gene expression in the adult mouse; rather it is predominantly the expression pattern of adipose tissue of the developing mouse between birth and weaning. The variation in gene expression among fat depots in both human and rodent has been an extensively studied phenomenon, and as recently reviewed, it is related to subphenotypes associated with immune function, the inflammatory response, fat depot blood flow, and insulin sensitivity. We suggest that adipose tissue biology in the period from birth to weaning is not just a staging platform for the emergence of adult white fat but that it has properties to serve the unique needs of energy metabolism in the newborn. A case in point is the differentiation of brite cells that occurs during this period followed by their involution immediately following weaning.     

A Role for Bone Morphogenetic Protein-4 in Adipocyte Development

Obesity is characterized by increases in the number of mature adipocytes. Nascent adipocytes arise from mesenchymal stem cells (MSCs) by a multi-step process — MSCs are recruited to the adipocyte lineage forming determined preadipocytes, these committed progenitors proliferate, undergo growth arrest, and finally differentiate into mature adipocytes. Although the genetic mechanisms that control the differentiation of preadipocytes into mature adipocytes are understood to a large extent, the earliest events in adipogenesis — especially the commitment of MSCs into preadipocytes — are largely unknown. Recently, bone morphogenetic protein-4 (BMP-4) has been implicated in the commitment of pluripotent MSCs to the adipocyte lineage by two independent lines of investigation. First, growth-arrested 10T1/2 cells do not normally respond to a hormonal cocktail that causes various growth-arrested preadipocyte cell lines to differentiate into adipocytes, but if 10T1/2 cells are first treated with BMP-4 they will respond to these hormonal inducers by undergoing terminal adipocyte differentiation. Second, a preadipocyte cell line, A33 cells, derived from 10T1/2 cells after exposing the cells to the DNA methyltransferase inhibitor 5-azacytidine was shown to express BMP-4, and this endogenous BMP-4 expression is required for acquisition of the preadipocyte phenotype of these cells. A role for the BMP-4 signaling pathway in adipogenesis is discussed.     

Pluripotent stem cells: Maintenance of genetic and epigenetic stability and prospects of cell technologies

Permanent lines of pluripotent stem cells can be obtained from humans and monkeys using different techniques and from different sources—inner cell mass of the blastocyst, primary germ cells, parthenogenetic oocytes, and mature spermatogonia—as well as by transgenic modification of various adult somatic cells. Despite different origin, all pluripotent lines demonstrate considerable similarity of the major biological properties: active self-renewal and differentiation into various somatic and germ cells in vitro and in vivo, similar gene expression profiles, and similar cell cycle structure. Ten years of intense studies on the stability of different human and monkey embryonic stem cells demonstrated that, irrespective of their origin, long-term in vitro cultures lead to the accumulation of chromosomal and gene mutations as well as epigenetic changes that can cause oncogenic transformation of cells. This review summarizes the research data on the genetic and epigenetic stability of different lines of pluripotent stem cells after long-term in vitro culture. These data were used to analyze possible factors of the genome and epigenome instability in pluripotent lines. The prospects of using pluripotent stem cells of different origin in cell therapy and pharmacological studies were considered.