Human cells with osteogenic potential in bone tissue research.

Bone regeneration after injury or after surgical bone removal due to disease is a serious medical challenge. A variety of materials are being tested to replace a missing bone or tooth. Regeneration requires cells capable of proliferation and differentiation in bone tissue. Although there are many possible human cell types available for use as a model for each phase of this process, no cell type is ideal for each phase. Osteosarcoma cells are preferred for initial adhesion assays due to their easy cultivation and fast proliferation, but they are not suitable for subsequent differentiation testing due to their cancer origin and genetic differences from normal bone tissue. Mesenchymal stem cells are more suitable for biocompatibility testing, because they mimic natural conditions in healthy bone, but they proliferate more slowly, soon undergo senescence, and some subpopulations may exhibit weak osteodifferentiation. Primary human osteoblasts provide relevant results in evaluating the effect of biomaterials on cellular activity; however, their resources are limited for the same reasons, like for mesenchymal stem cells. This review article provides an overview of cell models for biocompatibility testing of materials used in bone tissue research.

Pluripotency Stemness and Cancer: More Questions than Answers.

Embryonic stem cells and induced pluripotent stem cells provided us with fascinating new knowledge in recent years. Mechanistic insight into intricate regulatory circuitry governing pluripotency stemness and disclosing parallels between pluripotency stemness and cancer instigated numerous studies focusing on roles of pluripotency transcription factors, including Oct4, Sox2, Klf4, Nanog, Sall4 and Tfcp2L1, in cancer. Although generally well substantiated as tumour-promoting factors, oncogenic roles of pluripotency transcription factors and their clinical impacts are revealing themselves as increasingly complex. In certain tumours, both Oct4 and Sox2 behave as genuine oncogenes, and reporter genes driven by composite regulatory elements jointly recognized by both the factors can identify stem-like cells in a proportion of tumours. On the other hand, cancer stem cells seem to be biologically very heterogeneous both among different tumour types and among and even within individual tumours. Pluripotency transcription factors are certainly implicated in cancer stemness, but do not seem to encompass its entire spectrum. Certain cancer stem cells maintain their stemness by biological mechanisms completely different from pluripotency stemness, sometimes even by engaging signalling pathways that promote differentiation of pluripotent stem cells. Moreover, while these signalling pathways may well be antithetical to stemness in pluripotent stem cells, they may cooperate with pluripotency factors in cancer stem cells - a paradigmatic example is provided by the MAPK-AP-1 pathway. Unexpectedly, forced expression of pluripotency transcription factors in cancer cells frequently results in loss of their tumour-initiating ability, their phenotypic reversion and partial epigenetic normalization. Besides the very different signalling contexts operating in pluripotent and cancer stem cells, respectively, the pronounced dose dependency of reprogramming pluripotency factors may also contribute to the frequent loss of tumorigenicity observed in induced pluripotent cancer cells. Finally, contradictory cell-autonomous and non-cell-autonomous effects of various signalling molecules operate during pluripotency (cancer) reprogramming. The effects of pluripotency transcription factors in cancer are thus best explained within the concept of cancer stem cell heterogeneity.

Chronic DDE Exposure Modifies Mitochondrial Respiration during Differentiation of Human Adipose-Derived Mesenchymal Stem Cells into Mature Adipocytes.

The contribution of environmental pollutants to the obesity pandemic is still not yet fully recognized. Elucidating possible cellular and molecular mechanisms of their effects is of high importance. Our study aimed to evaluate the effect of chronic, 21-day-long, 2,2-bis (4-chlorophenyl)-1,1-dichlorethylenedichlorodiphenyldichloroethylene (p,p'-DDE) exposure of human adipose-derived mesenchymal stem cells committed to adipogenesis on mitochondrial oxygen consumption on days 4, 10, and 21. In addition, the mitochondrial membrane potential (MMP), the quality of the mitochondrial network, and lipid accumulation in maturing cells were evaluated. Compared to control differentiating adipocytes, exposure to p,p'-DDE at 1 µM concentration significantly increased basal (routine) mitochondrial respiration, ATP-linked oxygen consumption and MMP of intact cells on day 21 of adipogenesis. In contrast, higher pollutant concentration seemed to slow down the gradual increase in ATP-linked oxygen consumption typical for normal adipogenesis. Organochlorine p,p'-DDE did not alter citrate synthase activity. In conclusion, in vitro 1 µM p,p'-DDE corresponding to human exposure is able to increase the mitochondrial respiration per individual mitochondrion at the end of adipocyte maturation. Our data reveal that long-lasting exposure to p,p'-DDE could interfere with the metabolic programming of mature adipocytes.

Complex Interplay of Genes Underlies Invasiveness in Fibrosarcoma Progression Model.

Sarcomas are a heterogeneous group of mesenchymal tumours, with a great variability in their clinical behaviour. While our knowledge of sarcoma initiation has advanced rapidly in recent years, relatively little is known about mechanisms of sarcoma progression. JUN-murine fibrosarcoma progression series consists of four sarcoma cell lines, JUN-1, JUN-2, JUN-2fos-3, and JUN-3. JUN-1 and -2 were established from a single tumour initiated in a H2K/v-jun transgenic mouse, JUN-3 originates from a different tumour in the same animal, and JUN-2fos-3 results from a targeted in vitro transformation of the JUN-2 cell line. The JUN-1, -2, and -3 cell lines represent a linear progression from the least transformed JUN-2 to the most transformed JUN-3, with regard to all the transformation characteristics studied, while the JUN-2fos-3 cell line exhibits a unique transformation mode, with little deregulation of cell growth and proliferation, but pronounced motility and invasiveness. The invasive sarcoma sublines JUN-2fos-3 and JUN-3 show complex metabolic profiles, with activation of both mitochondrial oxidative phosphorylation and glycolysis and a significant increase in spared respiratory capacity. The specific transcriptomic profile of invasive sublines features very complex biological relationships across the identified genes and proteins, with accentuated autocrine control of motility and angiogenesis. Pharmacologic inhibition of one of the autocrine motility factors identified, Ccl8, significantly diminished both motility and invasiveness of the highly transformed fibrosarcoma cell. This progression series could be greatly valuable for deciphering crucial aspects of sarcoma progression and defining new prognostic markers and potential therapeutic targets.

Urothelial Cancer Stem Cell Heterogeneity.

Urothelial carcinoma is a tumor type featuring pronounced intertumoral heterogeneity and a high mutational and epigenetic load. The two major histopathological urothelial carcinoma types - the non-muscle-invasive and muscle-invasive urothelial carcinoma - markedly differ in terms of their respective typical mutational profiles and also by their probable cells of origin, that is, a urothelial basal cell for muscle-invasive carcinomas and a urothelial intermediate cell for at least a large part of non-muscle-invasive carcinomas. Both non-muscle-invasive and muscle-invasive urothelial carcinomas can be further classified into discrete intrinsic subtypes based on their typical transcriptomic profiles. Urothelial carcinogenesis shows a number of parallels to a urothelial regenerative response. Both of these processes seem to be dominated by specific stem cell populations. In the last years, the nature and location of urothelial stem cell(s) have been subject to many controversies, which now seem to be settled down, favoring the existence of a largely single urothelial stem cell type located among basal cells. Basal cell markers have also been amply used to identify urothelial carcinoma stem cells, especially in muscle-invasive disease, but they proved useful even in some non-muscle-invasive tumors. Analyses on molecular nature of urothelial carcinoma stem cells performed till now point to their great heterogeneity, both during the tumor development and upon intertumoral comparison, sexual dimorphism providing a special example of the latter. Moreover, urothelial cancer stem cells are endowed with intrinsic plasticity, whereby they can modulate their stemness in relation to other tumor-related traits, especially motility and invasiveness. Such transitional modulations suggest underlying epigenetic mechanisms and, even within this context, inter- and intratumoral heterogeneity becomes apparent. Multiple molecular aspects of urothelial cancer stem cell biology markedly influence therapeutic response, implying their knowledge as a prerequisite to improved therapies of this disease. At the same time, the notion of urothelial cancer stem cell heterogeneity implies that this therapeutic benefit would be most probably and most efficiently achieved within the context of individualized antitumor therapy.

Ovarian Cancer Stem Cell Heterogeneity.

Ovarian carcinoma features pronounced clinical, histopathological, and molecular heterogeneity. There is good reason to believe that parts of this heterogeneity can be explained by differences in the respective cell of origin, with a self-renewing fallopian tube secretory cell being likely responsible for initiation of an overwhelming majority of high-grade serous ovarian carcinomas (i.e., type II tumors according to the recent dualistic classification), whereas there are several mutually non-exclusive possibilities for the initiation of type I tumors, including ovarian surface epithelium stem cells, endometrial cells, or even cells of extra-Müllerian origin. Interestingly, both fallopian tube self-renewing secretory cells and ovarian surface epithelium stem cells seem to be characterized by an overlapping array of stemness signaling pathways, especially Wnt/ß-catenin. Apart from this variability in the respective cell of origin, the particular clinical behavior of ovarian carcinoma strongly suggests an underlying stem cell component with a crucial impact. This becomes especially evident in high-grade serous ovarian carcinomas treated with classical chemotherapy, which entails a gradual evolution of chemoresistant disease without any apparent selection of clones carrying obvious chemoresistance-associated mutations. Several cell surface markers (e.g., CD24, CD44, CD117, CD133, and ROR1) as well as functional approaches (ALDEFLUOR™ and side population assays) have been used to identify and characterize putative ovarian carcinoma stem cells. We have recently shown that side population cells exhibit marked heterogeneity on their own, which can hamper their straightforward therapeutic targeting. An alternative strategy for stemness-depleting interventions is to target the stem cell niche, i.e., the specific microanatomical structure that secures stem cell maintenance and survival through provision of a set of stem cell-promoting and differentiation-antagonizing factors. Besides identifying direct or indirect therapeutic targets, profiling of side population cells and other ovarian carcinoma stem cell subpopulations can reveal relevant prognostic markers, as exemplified by our recent discovery of the Vav3.1 transcript variant, which filters out a fraction of prognostically unfavorable ovarian carcinoma cases.

Sarcoma Stem Cell Heterogeneity.

Sarcomas represent an extensive group of divergent malignant diseases, with the only common characteristic of being derived from mesenchymal cells. As such, sarcomas are by definition very heterogeneous, and this heterogeneity does not manifest only upon intertumoral comparison on a bulk tumor level but can be extended to intratumoral level. Whereas part of this intratumoral heterogeneity could be understood in terms of clonal genetic evolution, an essential part includes a hierarchical relationship between sarcoma cells, governed by both genetic and epigenetic influences, signals that sarcoma cells are exposed to, and intrinsic developmental programs derived from sarcoma cells of origin. The notion of this functional hierarchy operating within each tumor implies the existence of sarcoma stem cells, which may originate from mesenchymal stem cells, and indeed, mesenchymal stem cells have been used to establish several crucial experimental sarcoma models and to trace down their respective stem cell populations. Mesenchymal stem cells themselves are heterogeneous, and, moreover, there are alternative possibilities for sarcoma cells of origin, like neural crest-derived stem cells, or mesenchymal committed precursor cells, or - in rhabdomyosarcoma - muscle satellite cells. These various origins result in substantial heterogeneity in possible sarcoma initiation. Genetic and epigenetic changes associated with sarcomagenesis profoundly impact the biology of sarcoma stem cells. For pediatric sarcomas featuring discrete reciprocal translocations and largely stable karyotypes, the translocation-activated oncogenes could be crucial factors that confer stemness, principally by modifying transcriptome and interfering with normal epigenetic regulation; the most extensively studied examples of this process are myxoid/round cell liposarcoma, Ewing sarcoma, and synovial sarcoma. For adult sarcomas, which have typically complex and unstable karyotypes, stemness might be defined more operationally, as a reflection of actual assembly of genetically and epigenetically conditioned stemness factors, with dedifferentiated liposarcoma providing a most thoroughly studied example. Alternatively, stemness can be imposed by tumor microenvironment, as extensively documented in osteosarcoma. In spite of this heterogeneity in both sarcoma initiation and underlying stemness biology, some of the molecular mechanisms of stemness might be remarkably similar in diverse sarcoma types, like abrogation of classical tumor suppressors pRb and p53, activation of Sox-2, or inhibition of canonical Wnt/ß-catenin signaling. Moreover, even some stem cell markers initially characterized for their stem cell enrichment capacity in various carcinomas or leukemias seem to function quite similarly in various sarcomas. Understanding the biology of sarcoma stem cells could significantly improve sarcoma patient clinical care, leading to both better patient stratification and, hopefully, development of more effective therapeutic options.

Urothelial Carcinoma Stem Cells: Current Concepts, Controversies, and Methods.

Cancer stem cells are defined as a self-renewing and self-protecting subpopulation of cancer cells able to differentiate into morphologically and functionally diverse cancer cells with a limited lifespan. To purify cancer stem cells, two basic approaches can be applied, the marker-based approach employing various more of less-specific cell surface marker molecules and a marker-free approach largely based on various self-protection mechanisms. Within the context of urothelial carcinoma, both methods could find use. The cell surface markers have been mainly derived from the urothelial basal cell, a probable cell of origin of muscle-invasive urothelial carcinoma, with CD14, CD44, CD90, and 67LR representing successful examples of this strategy. The marker-free approaches involve side population sorting, for which a detailed protocol is provided, as well as the Aldefluor assay, which rely on a specific overexpression of efflux pumps or the detoxification enzyme aldehyde dehydrogenase, respectively, in stem cells. These assays have been applied to both non-muscle-invasive and muscle-invasive bladder cancer samples and cell lines. Urothelial carcinoma stem cells feature a pronounced heterogeneity as to their molecular stemness mechanisms. Several aspects of urothelial cancer stem cell biology could enter translational development rather soon, e.g., a specific CD44+-derived gene expression signature able to identify non-muscle-invasive bladder cancer patients with a high risk of progression, or deciphering a mechanism responsible for repopulating activity of urothelial carcinoma stem cells within the context of therapeutic resistance.

Prognostic Significance of TMPRSS2-ERG Fusion Gene in Prostate Cancer.

BACKGROUND/AIM: Current research of prostate cancer (PCa) offers a promising way of identifying patients with adverse prognosis who do benefit from radical treatment that can affect quality of life as resections are associated with numerous side-effects. The aim of our study was to evaluate the relationship of TMPRSS2-ERG fusion gene status, tumor tissue prostate-specific antigen (PSA), prostate cancer antigen 3 (PCA3), miR-23b, miR-26a and miR-221 expression levels in combination with preoperative serum PSA level to the risk of PCa recurrence after radical prostatectomy. PATIENTS AND METHODS: The study group consisted of 108 patients who underwent radical prostatectomy. PSA was measured in peripheral blood collected preoperativelly. The expression of TMPRSS2-ERG transcript and the expression of miR-23b, miR-26a and miR-221 in formalin-fixed, paraffin-embedded (FFPE) tumor tissues was analyzed by reverse transcription (RT) real-time polymerase chain reaction (PCR). RESULTS: Significantly shorter time to recurrence was observed in patients with high expression of TMPRSS2-ERG (p=0.0020). High levels of preoperative PSA (>10.0 ng/ml) proved to be marker of shorter time to recurrence (p=0.0153). The most promising marker of the risk of recurrence after radical prostatectomy was a combination of high level of preoperative serum PSA and high expression of TMPRSS2-ERG fusion transcript in tumor tissue (p=0.0001). CONCLUSION: A combination of high preoperative serum PSA and high expression of TMPRSS2-ERG could be promising in distinguishing those tumors that are aggressive and life-threatening.

Mitochondria in White, Brown, and Beige Adipocytes.

Mitochondria play a key role in energy metabolism in many tissues, including cardiac and skeletal muscle, brain, liver, and adipose tissue. Three types of adipose depots can be identified in mammals, commonly classified according to their colour appearance: the white (WAT), the brown (BAT), and the beige/brite/brown-like (bAT) adipose tissues. WAT is mainly involved in the storage and mobilization of energy and BAT is predominantly responsible for nonshivering thermogenesis. Recent data suggest that adipocyte mitochondria might play an important role in the development of obesity through defects in mitochondrial lipogenesis and lipolysis, regulation of adipocyte differentiation, apoptosis, production of oxygen radicals, efficiency of oxidative phosphorylation, and regulation of conversion of white adipocytes into brown-like adipocytes. This review summarizes the main characteristics of each adipose tissue subtype and describes morphological and functional modifications focusing on mitochondria and their activity in healthy and unhealthy adipocytes.