Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

The dystrophin gene, located at Xp21, codifies dystrophin, which is part of a protein complex responsible for the membrane stability of muscle cells. Its absence on muscle causes Duchenne Muscular Dystrophy (DMD), a severe disorder, while a defect of muscle dystrophin causes Becker Muscular Dystrophy (DMB), a milder disease. The replacement of the defective muscle through stem cells transplantation is a possible future treatment for these patients. Our objective was to analyze the potential of CD34+ stem cells from umbilical cord blood to differentiate in muscle cells and express dystrophin, in vitro. Protein expression was analyzed by Immunofluorescence, Western Blotting (WB) and Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR). CD34+ stem cells and myoblasts from a DMD affected patient started to fuse with muscle cells immediately after co-cultures establishment. Differentiation in mature myotubes was observed after 15 days and dystrophin-positive regions were detected through Immunofluorescence analysis. However, WB or RT-PCR analysis did not detect the presence of normal dystrophin in co-cultures of CD34+ and DMD or DMB affected patients' muscle cells. In contrast, some CD34+ stem cells differentiated in dystrophin producers' muscle cells, what was observed by WB, reinforcing that this progenitor cell has the potential to originate muscle dystrophin in vitro, and not just in vivo like reported before.

Differential distribution of stem cells in the auditory and vestibular organs of the inner ear

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.

LOW REACTIVE OXYGEN SPECIES AND HIGH GLYCOLYSIS IN GLIOBLASTOMA STEM CELLS:MECHANISMS AND THERAPEUTIC IMPLICATIONS

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of glioblastoma stem cells (GSCs), which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. Exposure of GSCs to fetal bovine serum (FBS) can cause their differentiation, the underlying mechanism of which remains unknown. Reactive oxygen species (ROS) play an important role in normal stem cell differentiation, but their role in affecting cancer stem cell fate remains unclear. Whether the metabolic characteristics of GSCs are different from other glioblastoma cells and can be targeted are also unknown. In this study, we used several stem-like glioblastoma cell lines derived from clinical tissues by typical neurosphere culture system or orthotopic xenografts, and showed that addition of fetal bovine serum to the medium induced an increase of ROS, leading to aberrant differentiation and decreases of stem cell markers such as CD133. We found that exposure of GSCs to serum induced their differentiation through activation of mitochondrial respiration, leading to an increase in superoxide (O2-) generation and a profound ROS stress response manifested by upregulation of oxidative stress response pathway. This increase in mitochondrial ROS led to a down-regulation of molecules including SOX2, and Olig2, and Notch1 that are important for stem cell function and an upregulation of mitochondrial superoxide dismutase SOD2 that converts O2- to H2O2. Neutralization of ROS by antioxidant N-acetyl-cysteine in the serum-treated GSCs suppressed the increase of superoxide and partially rescued the expression of SOX2, Olig2, and Notch1, and prevented the serum-induced differentiation phenotype. Additionally, GSCs showed high dependence on glycolysis for energy production. The combination of a glycolytic inhibitor 3-BrOP and a chemotherapeutic agent BCNU depleted cellular ATP and inhibited the repair of BCNU-induced DNA damage, achieving strikingly synergistic killing effects in drug resistant GSCs. This study uncovers the metabolic properties of glioblastoma stem cells and suggests that mitochondrial function and cellular redox status may profoundly affect the fates of glioblastoma stem cells via a ROS-mediated mechanism, and that the active glycolytic metabolism in cancer stem cells may provide a biochemical basis for developing novel therapeutic strategies to effectively eliminate GSCs.

Establishing guidelines for obtaining optimal cell sources to use in tendon tissue engineering strategies

Cell-based approaches in tissue engineering (TE) have been barely explored for the treatment of tendon and ligament (T/L) tissues, requiring the establishment of a widely available cell source with tenogenic potential. As T/L cells are scarce, stem cells may provide a good alternative. Understanding how resident cells behave in vitro, might be useful for recapitulating the tenogenic potential of stem cells for tendon TE applications. Therefore, we propose to isolate and characterize human T/L-derived cells (hTDCs and hLDCs) and compare their regenerative potential with stem cells from adipose tissue (hASCs) and amniotic fluid (hAFSCs)(1). T/L cells were isolated using different procedures and stem cells isolated as described elsewhere(1). Moreover, T/L cells were stimu- lated into the three mesenchymal lineages, using standard differentia- tion media. Cells were characterized for the typical stem cell markers as well as T/L related markers, namely tenascin-C, collagen I and III, decorin and scleraxis, using different complementary techniques such as real time RT-PCR, immunocytochemistry and flow cytometry. No differences were observed between T/L in gene expression and protein deposition. T/L cells were mostly positive for stem ness markers (CD73/CD90/CD105), and have the potential to differentiate towards osteogenesis, chondrogenesis and adipogenesis, demonstrated by the positive staining for AlizarinRed, SafraninO, ToluidineBlue and OilRed. hASCs and hAFSCs exhibit positive expression of all tenogenic mark- ers, although at lower levels than hTDCs and hLDCs. Nevertheless, stem cells availability is key factor in TE strategies, despite that it’s still required optimization to direct their tenogenic phenotype.

Inducible gene and shRNA expression in resident hematopoietic stem cells in vivo.

Hematopoietic stem cells (HSC) are probably the best understood somatic stem cells and often serve as a paradigm for other stem cells. Nevertheless, most current techniques to genetically manipulate them in vivo are either constitutive and/or induced in settings of hematopoietic stress such as after irradiation. Here, we present a conditional expression system that allows for externally controllable transgenesis and knockdown in resident HSCs, based on a lentiviral vector containing a tet-O sequence and a transgenic mouse line expressing a doxycyclin-regulated tTR-KRAB repressor protein. HSCs harvested from tTR-KRAB mice are transduced with the lentiviral vector containing a cDNA (i.e., Green Fluorescent Protein (GFP)) and/or shRNA (i.e., p53) of interest and then transplanted into lethally irradiated recipients. While the vector is effectively repressed by tTR-KRAB during homing and engraftment, robust GFP/shp53 expression is induced on doxycyclin treatment in HSCs and their progeny. Doxycylin-controllable transcription is maintained on serial transplantation, indicating that repopulating HSCs are stably modified by this approach. In summary, this easy to implement conditional system provides inducible and reversible overexpression or knock down of genes in resident HSCs in vivo using a drug devoid of toxic or activating effects.

Cardiac repair with allogeneic mesenchymal stem cells after myocardial infarction.

Over the past decade, use of autologous bone marrow-derived mononuclear cells (BMCs) has proven to be safe in phase-I/II studies in patients with myocardial infarction (MI). Taken as a whole, results support a modest yet significant improvement in cardiac function in cell-treated patients. Skeletal myoblasts, adipose-derived stem cells, and bone marrow-derived mesenchymal stem cells (MSCs) have also been tested in clinical studies. MSCs expand rapidly in vitro and have a potential for multilineage differentiation. However, their regenerative capacity decreases with aging, limiting efficacy in old patients. Allogeneic MSCs offer several advantages over autologous BMCs; however, immune rejection of allogeneic cells remains a key issue. As human MSCs do not express the human leukocyte antigen (HLA) class II under normal conditions, and because they modulate T-cell-mediated responses, it has been proposed that allogeneic MSCs may escape immunosurveillance. However, recent data suggest that allogeneic MSCs may switch immune states in vivo to express HLA class II, present alloantigen and induce immune rejection. Allogeneic MSCs, unlike syngeneic ones, were eliminated from rat hearts by 5 weeks, with a loss of functional benefit. Allogeneic MSCs have also been tested in initial clinical studies in cardiology patients. Intravenous allogeneic MSC infusion has proven to be safe in a phase-I trial in patients with acute MI. Endoventricular allogeneic MSC injection has been associated with reduced adverse cardiac events in a phase-II trial in patients with chronic heart failure. The long-term safety and efficacy of allogeneic MSCs for cardiac repair remain to be established. Ongoing phase-II trials are addressing these issues.

Gene expression profiling of rat spermatogonia and Sertoli cells reveals signaling pathways from stem cells to niche and testicular cancer cells to surrounding stroma.

Background: Stem cells and their niches are studied in many systems, but mammalian germ stem cells (GSC) and their niches are still poorly understood. In rat testis, spermatogonia and undifferentiated Sertoli cells proliferate before puberty, but at puberty most spermatogonia enter spermatogenesis, and Sertoli cells differentiate to support this program. Thus, pre-pubertal spermatogonia might possess GSC potential and pre-pubertal Sertoli cells niche functions. We hypothesized that the different stem cell pools at pre-puberty and maturity provide a model for the identification of stem cell and niche-specific genes. We compared the transcript profiles of spermatogonia and Sertoli cells from pre-pubertal and pubertal rats and examined how these related to genes expressed in testicular cancers, which might originate from inappropriate communication between GSCs and Sertoli cells. Results: The pre-pubertal spermatogonia-specific gene set comprised known stem cell and spermatogonial stem cell (SSC) markers. Similarly, the pre-pubertal Sertoli cell-specific gene set comprised known niche gene transcripts. A large fraction of these specifically enriched transcripts encoded trans-membrane, extra-cellular, and secreted proteins highlighting stem cell to niche communication. Comparing selective gene sets established in this study with published gene expression data of testicular cancers and their stroma, we identified sets expressed genes shared between testicular tumors and pre-pubertal spermatogonia, and tumor stroma and pre-pubertal Sertoli cells with statistic significance. Conclusions: Our data suggest that SSC and their niche specifically express complementary factors for cell communication and that the same factors might be implicated in the communication between tumor cells and their micro-enviroment in testicular cancer.

Phenotypic and functional analysis of human fetal liver hematopoietic stem cells in culture.

Steady-state hematopoiesis and hematopoietic transplantation rely on the unique potential of stem cells to undergo both self-renewal and multilineage differentiation. Fetal liver (FL) represents a promising alternative source of hematopoietic stem cells (HSCs), but limited by the total cell number obtained in a typical harvest. We reported that human FL nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) could be expanded under simple stroma-free culture conditions. Here, we sought to further characterize FL HSC/SRCs phenotypically and functionally before and following culture. Unexpanded or cultured FL cell suspensions were separated into various subpopulations. These were tested for long-term culture potential and for in vivo repopulating function following transplantation into NOD/SCID mice. We found that upon culture of human FL cells, a tight association between classical stem cell phenotypes, such as CD34(+) /CD38(-) and/or side population, and NOD/SCID repopulating function was lost, as observed with other sources. Although SRC activity before and following culture consistently correlated with the presence of a CD34(+) cell population, we provide evidence that, contrary to umbilical cord blood and adult sources, stem cells present in both CD34(+) and CD34(-) FL populations can sustain long-term hematopoietic cultures. Furthermore, upon additional culture, CD34-depleted cell suspensions, devoid of SRCs, regenerated a population of CD34(+) cells possessing SRC function. Our studies suggest that compared to neonatal and adult sources, the phenotypical characteristics of putative human FL HSCs may be less strictly defined, and reinforce the accumulated evidence that human FL represents a unique, valuable alternative and highly proliferative source of HSCs for clinical applications.

Interactions of diploid embryonal carcinoma cells and normal cell mass cells with 2.5 day mouse embryos following aggregation

Spontaneous teratocarcinomas are ovarian or testicular tumors which have their origins in germ cells. The tumors contain a disorganized array of benign differentiated cells as well as an undifferentiated population of malignant stem cells, the embryonal carcinoma or EC cells. These pluripotent stem cells in tissue culture share many properties with the transient pluripotent cells of the early embryo, and might therefore serve as models for the investigation of developmental events ill vitro. The property of EC cells of prime interest in this study is an in vivo phenomenon. Certain EC cell lines are known to be regulated ill vivo and to differentiate normally in association with normal embryonic cells, resulting in chimeric mice. These mice have two genetically distinct cell populations, one of which is derived from the originally malignant EC cells. This has usually been accomplished by injection of the EC cells into the Day 3 blastocyst. In this study, the interactions between earlier stage embryos and EC cells have been tested by aggregating clumps of EC cells with Day 2 embryos. The few previous aggregation studies produced a high degree of abnormality in chimeric embryos, but the EC cells employed had known chromosomal abnormalities. In this study, two diploid EC cell lines (P19 and Pi0) were aggregated with 2.5 day mouse embryos, and were found to behave quite differently in the embryonic environment. P19 containing aggregates generally resorbed early, and the few embryos recovered at midgestation were normal and non-chimeric. Pi0 containing aggregates survived in high numbers to midgestation, and the Pi0 cells were very successful in colonizing the embryo. All these embryos were chimeric, and the contribution by the EC cells to each chimera was very high. However, these heavily chimeric embryos were all abnormal. Blastocyst injection had previously produced some abnormal embryos with high Pl0 contributions in addition to the live born mice, which had lower EC contributions. This study now adds more support to the hypothesis that high EC contributions may be incompatible with normal development. The possibility that the abnormalities were due to the mixing of temporally asynchronous embryonic cell types in the aggregates was tested by aggregating normal pluripotent cells taken from 3.5 day embryos with 2.5 day embryos. Early embryo loss was very high, and histological studies showed that the majority of these embryos died by 6.5 days development. Some embryos escaped this early death such that some healthy chimeras were recovered, in contrast to recovery of abnormal chimeric embryos following Pl0-morula aggregations, and non-chimeric embryos following P19-morula aggregations. This somewhat surprising adverse effect on development following aggregation of normal cell types suggests that there are developmental difficulties associated with the mixing of asynchronous cell types in aggregates. However, the greater magnitude of the adverse effects when the aggregates contained tumor derived cells suggests that EC cells should not be considered the complete equivalent of the pluripotent cells of the early embryo.

Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal surgery

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.

Neural stem cells adopt tumorigenic properties by constitutively activated NF-kappaB and subsequent VEGF up-regulation

One of the challenges in stem cell research is to avoid transformation during cultivation. We studied high passage subventricular zone derived neural stem cells (NSCs) cultures of adult rats in the absence of growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We termed this culture exogenous growth factor independent neural stem cells (GiNSCs). GiNSCs expressed stemness markers, displayed a high constitutive NF-kappaB activity and an increased, aberrant, polyploid DNA content. GiNSCs showed a tumorigenic phenotype and formed colonies in a soft agar assay. Microarray analysis showed the up-regulation of the NF-kappaB target gene vascular endothelial growth factor (VEGF). In contrast, proneuronal genes were down-regulated. Under neuronal differentiation conditions GiNSCs adopted a glioma-like phenotype, with nuclear p53, preserving high amounts of Nestin positive cells and prolonged proliferation. Neutralization of VEGF strongly inhibited proliferation and induced differentiation. In a gain of function approach, the transfection of NSCs with constitutively active upstream kinase IKK-2 led to constitutively activated NF-kappaB, proliferation in absence of growth factors and augmented VEGF secretion. In a rescue experiment a reduction of NF-kappaB activity by overexpression of IkappaB-AA1 was able to shift the morphology toward an elongated cell form, increased cell death, and decreased proliferation. Thus GiNSCs may provide a potent tool in cancer research, as their exogenous cytokine independent proliferation and their constitutively high NF-kappaB expression presumes cancerous properties observed in gliomas. In addition, this study might add a novel mechanism for detecting oncogenic transformation in therapeutic stem cell cultures.

Adult craniofacial stem cells: sources and relation to the neural crest

During the process of development, neural crest cells migrate out from their niche between the newly formed ectoderm and the neural tube. Thereafter, they give rise not only to ectodermal cell types, but also to mesodermal cell types. Cell types with neural crest ancestry consequently comprise a number of specialized varieties, such as ectodermal neurons, melanocytes and Schwann cells, as well as mesodermal osteoblasts, adipocytes and smooth muscle cells. Numerous recent studies suggest that stem cells with a neural crest origin persist into adulthood, especially within the mammalian craniofacial compartment. This review discusses the sources of adult neural crest-derived stem cells (NCSCs) derived from the cranium, as well as their differentiation potential and expression of key stem cell markers. Furthermore, the expression of marker genes associated with embryonic stem cells and the issue of multi- versus pluripotency of adult NCSCs is reviewed. Stringent tests are proposed, which, if performed, are anticipated to clarify the issue of adult NCSC potency. Finally, current pre-clinical and clinical data are discussed in light of the clinical impact of adult NCSCs.

Enhanced Neural Progenitor/Stem Cells Self-Renewal via the Interaction of Stress-Inducible Protein 1 with the Prion Protein

Prion protein (PrPC), when associated with the secreted form of the stress-inducible protein 1 (STI1), plays an important role in neural survival, neuritogenesis, and memory formation. However, the role of the PrP(C)-STI1 complex in the physiology of neural progenitor/stem cells is unknown. In this article, we observed that neurospheres cultured from fetal forebrain of wild-type (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were maintained for several passages without the loss of self-renewal or multipotentiality, as assessed by their continued capacity to generate neurons, astrocytes, and oligodendrocytes. The homogeneous expression and colocalization of STI1 and PrP(C) suggest that they may associate and function as a complex in neurosphere-derived stem cells. The formation of neurospheres from Prnp(0/0) mice was reduced significantly when compared with their wild-type counterparts. In addition, blockade of secreted STI1, and its cell surface ligand, PrP(C), with specific antibodies, impaired Prnp(+/+) neurosphere formation without further impairing the formation of Prnp(0/0) neurospheres. Alternatively, neurosphere formation was enhanced by recombinant STI1 application in cells expressing PrP(C) but not in cells from Prnp(0/0) mice. The STI1-PrP(C) interaction was able to stimulate cell proliferation in the neurosphere-forming assay, while no effect on cell survival or the expression of neural markers was observed. These data suggest that the STI1-PrP(C) complex may play a critical role in neural progenitor/stem cells self-renewal via the modulation of cell proliferation, leading to the control of the stemness capacity of these cells during nervous system development. STEM CELLS 2011;29:1126-1136

Low power laser radiation at 685 nm stimulates stem-cell proliferation rate in Dugesia tigrina during regeneration

Today's scientific interest in tissue engineering for organ transplantations and regeneration from stem cells, allied with recent observations on biostimulation of tissues and cells by laser radiation, stands as a strong motivation for the present work, in which we examine the effects of the low power laser radiation onto planarians under regenerative process. To investigate those effects, a number of 60 amputated worms were divided in three study groups: a control group and two other groups submitted to daily 1 and 3 min long laser treatment sections at similar to 910 W/m(2) power density. A 685 nm diode laser with 35 mW optical power was used. Samples were sent to histological analysis at the 4th, the 7th and the 15th (lays after amputation. A remarkable increase in stem cells counts for the fourth day of regeneration was observed when the regenerating worms was stimulated by the laser radiation. Our findings encourage further research works on the influence of optical radiation onto stem cells and tissue regeneration. (c) 2005 Elsevier B.V. All rights reserved.

Increased Levels of NOTCH1, NF-kappa B, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)