Showing papers in "Stem Cells in 2010"

Mesenchymal Stromal Cells: Current Understanding and Clinical Status†

Abstract: Multipotent mesenchymal stromal cells (MSCs) represent a rare heterogeneous subset of pluripotent stromal cells that can be isolated from many different adult tissues that exhibit the potential to give rise to cells of diverse lineages. Numerous studies have reported beneficial effects of MSCs in tissue repair and regeneration. After culture expansion and in vivo administration, MSCs home to and engraft to injured tissues and modulate the inflammatory response through synergistic downregulation of proinflammatory cytokines and upregulation of both prosurvival and antiinflammatory factors. In addition, MSCs possess remarkable immunosuppressive properties, suppressing T-cell, NK cell functions, and also modulating dentritic cell activities. Tremendous progress has been made in preclinical studies using MSCs, including the ability to use allogeneic cells, which has driven the application of MSCs toward the clinical setting. This review highlights our current understanding into the biology of MSCs with particular emphasis on the cardiovascular and renal applications, and provides a brief update on the clinical status of MSC-based therapy.

A long-term follow-up study of intravenous autologous mesenchymal stem cell transplantation in patients with ischemic stroke.

Abstract: We previously evaluated the short-term follow-up preliminary data of mesenchymal stem cells (MSCs) transplantation in patients with ischemic stroke. The present study was conducted to evaluate the long-term safety and efficacy of i.v. MSCs transplantation in a larger population. To accomplish this, we performed an open-label, observer-blinded clinical trial of 85 patients with severe middle cerebral artery territory infarct. Patients were randomly allocated to one of two groups, those who received i.v. autologous ex vivo cultured MSCs (MSC group) or those who did not (control group), and followed for up to 5 years. Mortality of any cause, long-term side effects, and new-onset comorbidities were monitored. Of the 52 patients who were finally included in this study, 16 were the MSC group and 36 were the control group. Four (25%) patients in the MSC group and 21 (58.3%) in the control group died during the follow-up period, and the cumulative surviving portion at 260 weeks was 0.72 in the MSC group and 0.34 in the control group (log-rank; p = .058). Significant side effects were not observed following MSC treatment. The occurrence of comorbidities including seizures and recurrent vascular episodes did not differ between groups. When compared with the control group, the follow-up modified Rankin Scale (mRS) score was decreased, whereas the number of patients with a mRS of 0-3 increased in the MSC group (p = .046). Clinical improvement in the MSC group was associated with serum levels of stromal cell-derived factor-1 and the degree of involvement of the subventricular region of the lateral ventricle. Intravenous autologous MSCs transplantation was safe for stroke patients during long-term follow-up. This therapy may improve recovery after stroke depending on the specific characteristics of the patients.

Antibacterial Effect of Human Mesenchymal Stem Cells Is Mediated in Part from Secretion of the Antimicrobial Peptide LL‐37

Abstract: Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram-negative bacteria was the human cathelicidin antimicrobial peptide, hCAP-18/LL-37. Both m-RNA and protein expression data showed that the expression of LL-37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony-forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL-37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC-treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)-treated mice. Human bone marrow-derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP-18/ LL-37.

NOTCH Pathway Blockade Depletes CD133‐Positive Glioblastoma Cells and Inhibits Growth of Tumor Neurospheres and Xenografts

Abstract: Cancer stem cells (CSCs) are thought to be critical for the engraftment and long-term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem-like cells with higher NOTCH activity. We therefore used low-passage and established GBM-derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by γ-secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug-impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem-like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem-like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas.

Notch promotes radioresistance of glioma stem cells

Abstract: Radiotherapy represents the most effective nonsurgical treatments for gliomas. However, gliomas are highly radioresistant and recurrence is nearly universal. Results from our laboratory and other groups suggest that cancer stem cells contribute to radioresistance in gliomas and breast cancers. The Notch pathway is critically implicated in stem cell fate determination and cancer. In this study, we show that inhibition of Notch pathway with γ-secretase inhibitors (GSIs) renders the glioma stem cells more sensitive to radiation at clinically relevant doses. GSIs enhance radiation-induced cell death and impair clonogenic survival of glioma stem cells but not non-stem glioma cells. Expression of the constitutively active intracellular domains of Notch1 or Notch2 protect glioma stem cells against radiation. Notch inhibition with GSIs does not alter the DNA damage response of glioma stem cells after radiation but rather reduces Akt activity and Mcl-1 levels. Finally, knockdown of Notch1 or Notch2 sensitizes glioma stem cells to radiation and impairs xenograft tumor formation. Taken together, our results suggest a critical role of Notch signaling to regulate radioresistance of glioma stem cells. Inhibition of Notch signaling holds promise to improve the efficiency of current radiotherapy in glioma treatment. STEM CELLS 2010;28:17–28

The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells.

Abstract: The ability of stem cells to propagate indefinitely is believed to occur via the fine modulation of pathways commonly involved in cellular senescence, including the telomerase, the p53, and the mitochondrial/oxidative stress pathways. Induced pluripotent stem cells (iPSCs) are a novel stem cell population obtained from somatic cells through forced expression of a set of genes normally expressed in embryonic stem cells (ESCs). These reprogrammed cells acquire self-renewal properties and appear almost undistinguishable from ESCs in terms of morphology, gene expression, and differentiation potential. Accordingly, iPSCs exhibit alterations of the senescence-related telomerase and p53 signaling pathways. However, although treatments with antioxidants have been recently shown to enhance cellular reprogramming, detailed information regarding the state of the mitochondrial/oxidative stress pathway in iPSCs is still lacking. Mitochondria undergo specific changes during organismal development and aging. Thus, addressing whether somatic mitochondria within iPSCs acquire ESC-like features or retain the phenotype of the parental cell is an unanswered but relevant question. Herein, we demonstrate that somatic mitochondria within human iPSCs revert to an immature ESC-like state with respect to organelle morphology and distribution, expression of nuclear factors involved in mitochondrial biogenesis, content of mitochondrial DNA, intracellular ATP level, oxidative damage, and lactate generation. Upon differentiation, mitochondria within iPSCs and ESCs exhibited analogous maturation and anaerobic-to-aerobic metabolic modifications. Overall, the data highlight that human iPSCs and ESCs, although not identical, share similar mitochondrial properties and suggest that cellular reprogramming can modulate the mitochondrial/oxidative stress pathway, thus inducing a rejuvenated state capable of escaping cellular senescence.

MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation.

Abstract: Differentiation of mesenchymal stem cells into a particular lineage is tightly regulated, and malfunction of this regulation could lead to pathological consequences. Patients with osteoporosis have increased adipocyte accumulation, but the mechanisms involved remain to be defined. In this study, we aimed to investigate if microRNAs regulate mesenchymal progenitor cells and bone marrow stromal cell (BMSC) differentiation through modulation of Runx2, a key transcription factor for osteogenesis. We found that miR-204 and its homolog miR-211 were expressed in mesenchymal progenitor cell lines and BMSCs and their expression was induced during adipocyte differentiation, whereas Runx2 protein expression was suppressed. Retroviral overexpression of miR-204 or transfection of miR-204 oligo decreased Runx2 protein levels and miR-204 inhibition significantly elevated Runx2 protein levels, suggesting that miR-204 acts as an endogenous attenuator of Runx2 in mesenchymal progenitor cells and BMSCs. Mutations of putative miR-204 binding sites upregulated the Runx2 3'-UTR reporter activity, suggesting that miR-204/211 bind to Runx2 3'-UTR. Perturbation of miR-204 resulted in altered differentiation fate of mesenchymal progenitor cells and BMSCs: osteoblast differentiation was inhibited and adipocyte differentiation was promoted when miR-204 was overexpressed in these cells, whereasosteogenesis was upregulated and adipocyte formation was impaired when miR-204 was inhibited. Together, our data demonstrated that miR-204/211 act as important endogenous negative regulators of Runx2, which inhibit osteogenesis and promote adipogenesis of mesenchymal progenitor cells and BMSCs.

Human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing.

Abstract: Increasing evidence has supported the important role of mesenchymal stem cells (MSCs) in wound healing, however, the underlying mechanism remains unclear. Recently, we have isolated a unique population of MSCs from human gingiva (GMSCs) with similar stem cell-like properties, immunosuppressive, and anti-inflammatory functions as human bone marrow-derived MSCs (BMSCs). We describe here the interplay between GMSCs and macrophages and the potential relevance in skin wound healing. When cocultured with GMSCs, macrophages acquired an anti-inflammatory M2 phenotype characterized by an increased expression of mannose receptor (MR; CD206) and secretory cytokines interleukin (IL)-10 and IL-6, a suppressed production of tumor necrosis factor (TNF)-α, and decreased ability to induce Th-17 cell expansion. In vivo, we demonstrated that systemically infused GMSCs could home to the wound site in a tight spatial interaction with host macrophages, promoted them toward M2 polarization, and significantly enhanced wound repair. Mechanistically, GMSC treatment mitigated local inflammation mediated by a suppressed infiltration of inflammatory cells and production of IL-6 and TNF-α, and an increased expression of IL-10. The GMSC-induced suppression of TNF-α secretion by macrophages appears to correlate with impaired activation of NFκB p50. These findings provide first evidence that GMSCs are capable to elicit M2 polarization of macrophages, which might contribute to a marked acceleration of wound healing.

Butyrate Greatly Enhances Derivation of Human Induced Pluripotent Stem Cells by Promoting Epigenetic Remodeling and the Expression of Pluripotency-Associated Genes

Abstract: We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming.

Generation of Transgene‐Free Lung Disease‐Specific Human Induced Pluripotent Stem Cells Using a Single Excisable Lentiviral Stem Cell Cassette

Abstract: The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step toward the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. Although several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed to achieve the widespread derivation of disease-specific iPSC from humans with inherited or degenerative diseases. Here, we report the use of a humanized version of a single lentiviral "stem cell cassette" vector to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either three or four reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration that on removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, α-1 antitrypsin deficiency-related emphysema, scleroderma, and sickle-cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia.

In Vitro High-Capacity Assay to Quantify the Clonal Heterogeneity in Trilineage Potential of Mesenchymal Stem Cells Reveals a Complex Hierarchy of Lineage Commitment

Abstract: In regenerative medicine, bone marrow is a promising source of mesenchymal stem cells (MSCs) for a broad range of cellular therapies. This research addresses a basic prerequisite to realize the therapeutic potential of MSCs by developing a novel high-capacity assay to quantify the clonal heterogeneity in potency that is inherent to MSC preparations. The assay utilizes a 96-well format to (1) classify MSCs according to colony-forming efficiency as a measure of proliferation capacity and trilineage potential to exhibit adipo-, chondro-, and osteogenesis as a measure of multipotency and (2) preserve a frozen template of MSC clones of known potency for future use. The heterogeneity in trilineage potential of normal bone marrow MSCs is more complex than previously reported: all eight possible categories of trilineage potential were detected. In this study, the average colony-forming efficiency of MSC preparations was 55-62%, and tripotent MSCs accounted for nearly 50% of the colony-forming cells. The multiple phenotypes detected in this study infer a more convoluted hierarchy of lineage commitment than described in the literature. Greater cell amplification, colony-forming efficiency, and colony diameter for tri- versus unipotent clones suggest that MSC proliferation may be a function of potency. CD146 may be a marker of multipotency, with approximately 2-fold difference in mean fluorescence intensity between tri- and unipotent clones. The significance of these findings is discussed in the context of the efficacy of MSC therapies. The in vitro assay described herein will likely have numerous applications given the importance of heterogeneity to the therapeutic potential of MSCs.

Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N-cadherin.

Abstract: Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into many cell types. Chondrogenesis is induced in hMSCs cultured as a micromass pellet to mimic cellular condensation during cartilage development, and exposed to transforming growth factor beta (TGFbeta). Interestingly, TGFbeta can also induce hMSC differentiation to smooth-muscle-like cell types, but it remains unclear what directs commitment between these two lineages. Our previous work revealed that cell shape regulates hMSC commitment between osteoblasts and adipocytes through RhoA signaling. Here we show that cell shape also confers a switch between chondrogenic and smooth muscle cell (SMC) fates. Adherent and well-spread hMSCs stimulated with TGF beta 3 upregulated SMC genes, whereas cells allowed to attach onto micropatterned substrates, but prevented from spreading and flattening, upregulated chondrogenic genes. Interestingly, cells undergoing SMC differentiation exhibited little change in RhoA, but significantly higher Rac1 activity than chondrogenic cells. Rac1 activation inhibited chondrogenesis and was necessary and sufficient for inducing SMC differentiation. Furthermore, TGF beta 3 and Rac1 signaling upregulated N-cadherin, which was required for SMC differentiation. These results demonstrate a chondrogenic-SMC fate decision mediated by cell shape, Rac1, and N-cadherin, and highlight the tight coupling between lineage commitment and the many changes in cell shape, cell-matrix adhesion, and cell-cell adhesion that occur during morphogenesis.

Concise review: hitting the right spot with mesenchymal stromal cells.

Abstract: Mesenchymal stromal cells or mesenchymal stem cells (MSCs) have captured considerable scientific and public interest because of their potential to limit physical and immune injury, to produce bioactive molecules and to regenerate tissues. MSCs are phenotypically heterogeneous and distinct subpopulations within MSC cultures are presumed to contribute to tissue repair and the modulation of allogeneic immune responses. As the first example of efficacy, clinical trials for prevention and treatment of graft-versus-host disease after hematopoietic cell transplantation show that MSCs can effectively treat human disease. The view of the mechanisms whereby MSCs function as immunomodulatory and reparative cells has evolved simultaneously. Initially, donor MSCs were thought to replace damaged cells in injured tissues of the recipient. More recently, however, it has become increasingly clear that even transient MSC engraftment may exert favorable effects through the secretion of cytokines and other paracrine factors, which engage and recruit recipient cells in productive tissue repair. Thus, an important reason to investigate MSCs in mechanistic preclinical models and in clinical trials with well-defined end points and controls is to better understand the therapeutic potential of these multifunctional cells. Here, we review the controversies and recent insights into MSC biology, the regulation of alloresponses by MSCs in preclinical models, as well as clinical experience with MSC infusions (Table 1) and the challenges of manufacturing a ready supply of highly defined transplantable MSCs.

Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence.

Abstract: Human induced pluripotent stem cells (hiPSC) have been shown to differentiate into a variety of replacement cell types. Detailed evaluation and comparison with their human embryonic stem cell (hESC) counterparts is critical for assessment of their therapeutic potential. Using established methods, we demonstrate here that hiPSCs are capable of generating hemangioblasts/blast cells (BCs), endothelial cells, and hematopoietic cells with phenotypic and morphologic characteristics similar to those derived from hESCs, but with a dramatic decreased efficiency. Furthermore, in distinct contrast with the hESC derivatives, functional differences were observed in BCs derived from hiPSCs, including significantly increased apoptosis, severely limited growth and expansion capability, and a substantially decreased hematopoietic colony-forming capability. After further differentiation into erythroid cells, >1,000-fold difference in expansion capability was observed in hiPSC-BCs versus hESC-BCs. Although endothelial cells derived from hiPSCs were capable of taking up acetylated low-density lipoprotein and forming capillary-vascular-like structures on Matrigel, these cells also demonstrated early cellular senescence (most of the endothelial cells senesced after one passage). Similarly, retinal pigmented epithelium cells derived from hiPSCs began senescing in the first passage. Before clinical application, it will be necessary to determine the cause and extent of such abnormalities and whether they also occur in hiPSCs generated using different reprogramming methods.

MicroRNA miR-137 regulates neuronal maturation by targeting ubiquitin ligase Mind Bomb-1

Abstract: The maturation of young neurons is regulated by complex mechanisms and dysregulation of this process is frequently found in neurodevepmental disorders. MicroRNAs have been implicated in several steps of neuronal maturation including dendritic and axonal growth, spine development, and synaptogenesis. We demonstrate that one brain-enriched microRNA, miR-137, has a significant role in regulating neuronal maturation. Overexpression of miR-137 inhibits dendritic morphogenesis, phenotypic maturation, and spine development both in brain and cultured primary neurons. On the other hand, a reduction in miR-137 had opposite effects. We further show that miR-137 targets the Mind bomb one (Mib1) protein through the conserved target site located in the 3' untranslated region of Mib1 messenger RNA. Mib1 is an ubiquitin ligase known to be important for neurodevelopment. We show that exogenously expressed Mib1 could partially rescue the phenotypes associated with miR-137 overexpression. These results demonstrate a novel miRNA-mediated mechanism involving miR-137 and Mib1 that function to regulate neuronal maturation and dendritic morphogenesis during development.

Radiation Resistance of Cancer Stem Cells: The 4 R's of Radiobiology Revisited

Abstract: There is compelling evidence that many solid cancers are organized hierarchically and contain a small population of cancer stem cells (CSCs). It seems reasonable to suggest that a cancer cure can be achieved only if this population is eliminated. Unfortunately, there is growing evidence that CSCs are inherently resistant to radiation, and perhaps other cancer therapies. In general, success or failure of standard clinical radiation treatment is determined by the 4 R's of radiobiology: repair of DNA damage, redistribution of cells in the cell cycle, repopulation, and reoxygenation of hypoxic tumor areas. We relate recent findings on CSCs to these four phenomena and discuss possible consequences.

Allogeneic periodontal ligament stem cell therapy for periodontitis in swine.

Abstract: Periodontitis is one of the most widespread infectious diseases in humans. It is the main cause of tooth loss and associated with a number of systemic diseases. Until now, there is no appropriate method for functional periodontal tissue regeneration. Here, we establish a novel approach of using allogeneic periodontal ligament stem cells (PDLSCs) sheet to curing periodontitis in a miniature pig periodontitis model. Significant periodontal tissue regeneration was achieved in both the autologous and the allogeneic PDLSCs transplantation group at 12 weeks post-PDLSCs transplantation. Based on clinical assessments, computed tomography (CT) scanning, and histological examination, there was no marked difference between the autologous and allogeneic PDLSCs transplantation groups. In addition, lack of immunological rejections in the animals that received the allogeneic PDLSCs transplantation was observed. Interestingly, we found that human PDLSCs fail to express human leukocyte antigen (HLA)-II DR and costimulatory molecules. PDLSCs were not able to elicit T-cell proliferation and inhibit T-cell proliferation when stimulated with mismatched major histocompatibility complex molecules. Furthermore, we found that prostaglandin E2 (PGE2) plays a crucial role in PDLSCs-mediated immunomodulation and periodontal tissue regeneration in vitro and in vivo. Our study demonstrated that PDLSCs possess low immunogenicity and marked immunosuppression via PGE2-induced T-cell anergy. We developed a standard technological procedure of using allogeneic PDLSCs to cure periodontitis in swine. Stem Cells 2010;28:1829–1838

Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Improve Recovery after Cervical Spinal Cord Injury

Abstract: Evidence that cell transplants can improve recovery outcomes in spinal cord injury (SCI) models substantiates treatment strategies involving cell replacement for humans with SCI. Most pre-clinical studies of cell replacement in SCI examine thoracic injury models. However, as most human injuries occur at the cervical level, it is critical to assess potential treatments in cervical injury models and examine their effectiveness using at-level histological and functional measures. To directly address cervical SCI, we used a C5 midline contusion injury model and assessed the efficacy of a candidate therapeutic for thoracic SCI in this cervical model. The contusion generates reproducible, bilateral movement and histological deficits, although a number of injury parameters such as acute severity of injury, affected gray-to-white matter ratio, extent of endogenous remyelination, and at-level locomotion deficits do not correspond with these parameters in thoracic SCI. On the basis of reported benefits in thoracic SCI, we transplanted human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells (OPCs) into this cervical model. hESC-derived OPC transplants attenuated lesion pathogenesis and improved recovery of forelimb function. Histological effects of transplantation included robust white and gray matter sparing at the injury epicenter and, in particular, preservation of motor neurons that correlated with movement recovery. These findings further our understanding of the histopathology and functional outcomes of cervical SCI, define potential therapeutic targets, and support the use of these cells as a treatment for cervical SCI.

Human Induced Pluripotent Stem Cells Develop Teratoma More Efficiently and Faster Than Human Embryonic Stem Cells Regardless the Site of Injection

Abstract: Human embryonic stem cell (hESC) and reprogrammed/induced pluripotent stem cell (iPSC) research is becoming the “flavor of the month” for downstream applications such as drug screening, disease modeling, and future regenerative medicine and cell therapies [1–4]. Pluripotency (the ability to give rise to any cell type of the three germ layers: mesoderm, ectoderm, and endoderm) is the defining feature of hESCs and iPSCs [5]. In vivo teratoma formation in immune-compromised mice is the “gold-standard” assay to define bona fide pluripotent stem cells capable of generating tumoral disorganized structures containing tissues representing the three germ layers [5,6]. Despite the importance of teratoma assay as an extended screen for the pluripotency of hESCs and iPSCs and as in vivo assay to explore molecular and cellular mechanisms underlying the biology of human teratomas and their transition to teratocarcinomas, there are no standard procedures for performing this assay [5–7]. Different studies on hESCs have correlated the site of implantation with the efficiency of teratoma formation and histology tissue composition [6,8]. However, limited data are available regarding the teratoma development latency. More importantly, no study so far has compared side-by-side the efficiency, latency, and histological tumor composition of hESCs- and iPSCs-derived teratomas. In addition, a new generation of immunodeficient mice has been developed: the NOD/SCID IL2Rγ−/− mouse. This strain carries a IL2Rγ-chain deficiency that blocks signaling through multiple cytokine receptors leading to many innate immune defects [9,10]. The non obese diabetic/severe combined immune-deficient (NOD/SCID) IL2Rγ−/− strain facilitates engraftment and tumor formation and does not develop thymic lymphoma, ensuring a longer lifespan of inoculated mice. Here, we followed the improved teratoma protocol previously developed by Prokhorova et al. [6,11–13] to transplant side-by-side as few as 1 × 106 of either fully characterized undifferentiated hESCs or iPSCs in 6- to 8-week-old non obese diabetic/severe combined immune-deficient (NOD/SCID) IL2Rγ−/− mice [11,13–15]. The following hESC lines were used: H9, H1, AND1, AND2, AND3, HS181, and ECAT. The following iPSC lines were used: MSHU-001, iAND4, CB-CD34+ iPSC1, and CB-CD34+ iPSC2. These lines have been fully characterized and deposited according to Spanish Legislation at The Spanish Stem Cell Bank (http://www.isciii.es/htdocs/terapia/terapia_lineas.jsp) [16]. Briefly, cells were resuspended in phosphate buffered saline (PBS) supplemented with 30% matrigel (Becton Dickinson, San Jose, CA, http://www.bd.com) [6] and transplanted subcutaneously (200 μl volume) or by intratesticular injection (60 μl volume). Figure ​Figure1A1A depicts the experimental strategy used. We then analyzed efficiency, latency, and histological tumor composition. In hESCs, the rate of teratoma formation was 81% subcutaneously versus 94% intratesticularly (n = 30 mice; Fig. ​Fig.1B).1B). However, the intratesticular injection, despite showing higher efficiency of teratoma formation, displayed a slightly longer latency (66 vs. 59 days; p-value > 0.05). There were no site-specific differences in the teratoma composition at the histological level (Fig. ​(Fig.1C).1C). Interestingly, when iPSCs were transplanted the rate of teratoma formation was 100% (n = 16 mice), regardless the type of injection. More importantly, iPSCs seem more aggressive in vivo as the latency was shortened 52% (from 59 days to 31 days) upon subcutaneous injection and 26% (from 66 days to 49 days) upon intratesticular injection. As with hESCs, no differences in teratoma composition were observed either. Figure 1 Human iPSCs form teratomas faster and with higher efficiency than hESCs regardless the site of injection. (A): Cartoon summarizing the experimental design. (B): Table summarizing the efficiency, latency, and histological analysis of the teratomas developed ... To the best of our knowledge, this is the first study comparing side-by-side the efficiency, latency, and teratoma composition between hESCs and iPSCs. We found clear differences in the efficiency and latency but not in the teratoma histological composition. Further experiments are still demanded to gain insights into the higher aggressiveness in vivo of iPSCs as compared with hESCs. Ploidy, analyzed by conventional G-banding karyotype, could not explained these differences because all but two pluripotent stem cell lines were euploid: the aneuploid lines were one hESC (AND1) and one iPSC (iAND4). It is worth emphasizing, however, that karyotype analysis is not a high-resolution technique detecting fine genomic aberrations, with a euploid karyotype not being therefore indicative of an overall cellular genomic stability. Whether or not specific tiny genomic insults (detectable by high-resolution methods such as comparative genomic hybridazation (CGH)-arrays and single-nucleotide polymorphism analysis) or epigenetic differences may explain the higher aggressiveness in vivo of iPSCs still needs to be elucidated. We envision that these data may be useful not only for stem cells scientists addressing pluripotency issues and studying mechanisms underlying specific germ-layer/tissue differentiation but also for cancer researchers developing in vivo models for germ cell tumors.

Efficient Generation of Functional Dopaminergic Neurons from Human Induced Pluripotent Stem Cells Under Defined Conditions

Abstract: Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells represent a promising unlimited cell source for generating patient-specific cells for biomedical research and personalized medicine. As a first step, critical to clinical applications, we attempted to develop defined culture conditions to expand and differentiate human iPSCs into functional progeny such as dopaminergic neurons for treating or modeling Parkinson's disease (PD). We used a completely defined (xeno-free) system that we previously developed for efficient generation of authentic dopaminergic neurons from human embryonic stem cells (hESCs), and applied it to iPSCs. First, we adapted two human iPSC lines derived from different somatic cell types for the defined expansion medium and showed that the iPSCs grew similarly as hESCs in the same medium regarding pluripotency and genomic stability. Second, by using these two independent adapted iPSC lines, we showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was also similar to hESCs. Importantly, iPSC-derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. In addition, iPSC-derived NSCs and neurons could be efficiently transduced by a baculoviral vector delivering episomal DNA for future gene function study and disease modeling using iPSCs. We also performed genome-wide microarray comparisons between iPSCs and hESCs, and we derived NSC and dopaminergic neurons. Our data revealed overall similarity and visible differences at a molecular level. Efficient generation of functional dopaminergic neurons under defined conditions will facilitate research and applications using PD patient-specific iPSCs.

Nestin is required for the proper self-renewal of neural stem cells.

Abstract: The intermediate filament protein, nestin, is a widely employed marker of multipotent neural stem cells (NSCs). Recent in vitro studies have implicated nestin in a number of cellular processes, but there is no data yet on its in vivo function. Here, we report the construction and functional characterization of Nestin knockout mice. We found that these mice show embryonic lethality, with neuroepithelium of the developing neural tube exhibiting significantly fewer NSCs and much higher levels of apoptosis. Consistent with this in vivo observation, NSC cultures derived from knockout embryos show dramatically reduced self-renewal ability that is associated with elevated apoptosis but no overt defects in cell proliferation or differentiation. Unexpectedly, nestin deficiency has no detectable effect on the integrity of the cytoskeleton. Furthermore, the knockout of Vimentin, which abolishes nestin's ability to polymerize into intermediate filaments in NSCs, does not lead to any apoptotic phenotype. These data demonstrate that nestin is important for the proper survival and self-renewal of NSCs, and that this function is surprisingly uncoupled from nestin's structural involvement in the cytoskeleton.

Excision of Reprogramming Transgenes Improves the Differentiation Potential of iPS Cells Generated with a Single Excisable Vector

Abstract: The residual presence of integrated transgenes following the derivation of induced pluripotent stem (iPS) cells is highly undesirable. Here we demonstrate efficient derivation of iPS cells free of exogenous reprogramming transgenes using an excisable polycistronic lentiviral vector. A novel version of this vector containing a reporter fluorochrome allows direct visualization of vector excision in living iPS cells in real time. We find that removal of the reprogramming vector markedly improves the developmental potential of iPS cells and significantly augments their capacity to undergo directed differentiation in vitro. We further propose that methods to efficiently excise reprogramming transgenes with minimal culture passaging, such as those demonstrated here, are critical since we find that iPS cells may acquire chromosomal abnormalities, such as trisomy of chromosome 8, similar to embryonic stem cells after expansion in culture. Our findings illustrate an efficient method for the generation of transgene-free iPS cells and emphasize the potential beneficial effects that may result from elimination of integrated reprogramming factors. In addition, our results underscore the consequences of long-term culture that will need to be taken into account for the clinical application of iPS cells.

Inhibition of Notch Signaling in Glioblastoma Targets Cancer Stem Cells via an Endothelial Cell Intermediate

Abstract: Glioblastoma multiforme (GBM) is a highly heterogeneous malignant tumor. Recent data suggests the presence of a hierarchical organization within the GBM cell population that involves cancer cells with stem-like behavior, capable of repopulating the tumor and contributing to its resistance to therapy. Tumor stem cells are thought to reside within a vascular niche that provides structural and functional support. However, most GBM studies involve isolated tumor cells grown under various culture conditions. Here, we use a novel three-dimensional organotypic "explant" system of surgical GBM specimens that preserves cytoarchitecture and tumor stroma along with tumor cells. Notch inhibition in explants results in decreased proliferation and self-renewal of tumor cells but is also associated with a decrease in endothelial cells. When endothelial cells are selectively eliminated from the explants via a toxin conjugate, we also observed a decrease in self-renewal of tumor stem cells. These findings support a critical role for tumor endothelial cells in GBM stem cell maintenance, mediated at least in part by Notch signaling. The explant system further highlighted differences in the response to radiation between explants and isolated tumor neurospheres. Combination treatment with Notch blockade and radiation resulted in a substantial decrease in proliferation and in self-renewal in tumor explants while radiation alone was less effective. This data suggests that the Notch pathway plays a critical role in linking angiogenesis and cancer stem cell self-renewal and is thus a potential therapeutic target. Three-dimensional explant systems provide a novel approach for the study of tumor and microenvironment interactions.

Intratumoral hypoxic gradient drives stem cells distribution and MGMT expression in glioblastoma.

Abstract: Glioblastoma multiforme (GBM) are highly proliferative tumors currently treated by surgical removal, followed by radiotherapy and chemotherapy, which are counteracted by intratumoral hypoxia. Here we exploited image guided surgery to sample multiple intratumoral areas to define potential cellular heterogeneity in correlation to the oxygen tension gradient within the GBM mass. Our results indicate that more immature cells are localized in the inner core and in the intermediate layer of the tumor mass, whereas more committed cells, expressing glial fibrillary acidic protein and beta-III-tubulin, are distributed along the peripheral and neo-vascularized area, where Smad1/5/8 and Stat3 result to be activated. Moreover, GBM stem cells, identified with the stem cell marker CD133, express high level of DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) known to be involved in chemotherapy resistance and highly expressed in the inner core of the tumor mass. Importantly, these cells and, particularly, CD133(+) cells result to be resistant to temozolomide (TMZ), the most used oral alkylating agent for the treatment of GBM, which specifically causes apoptosis only in GBM cells derived from the peripheral layer of the tumor mass. These results indicate a correlation between the intratumoral hypoxic gradient, the tumor cell phenotype, and the tumor resistance to chemotherapy leading to a novel concentric model of tumor stem cell niche, which may be useful to define the real localization of the chemoresistant GBM tumor cells in order to design more effective treatment strategies.

Intracerebral Transplantation of Bone Marrow‐Derived Mesenchymal Stem Cells Reduces Amyloid‐Beta Deposition and Rescues Memory Deficits in Alzheimer's Disease Mice by Modulation of Immune Responses

Abstract: Alzheimer's disease (AD) is characterized by the deposition of amyloid-beta peptide (Abeta) and the formation of neurofibrillary tangles. Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) has been suggested as a potential therapeutic approach to prevent various neurodegenerative disorders, including AD. However, the actual therapeutic impact of BM-MSCs and their mechanism of action in AD have not yet been ascertained. The aim of this study was therefore to evaluate the therapeutic effect of BM-MSC transplantation on the neuropathology and memory deficits in amyloid precursor protein (APP) and presenilin one (PS1) double-transgenic mice. Here we show that intracerebral transplantation of BM-MSCs into APP/PS1 mice significantly reduced amyloid beta-peptide (Abeta) deposition. Interestingly, these effects were associated with restoration of defective microglial function, as evidenced by increased Abeta-degrading factors, decreased inflammatory responses, and elevation of alternatively activated microglial markers. Furthermore, APP/PS1 mice treated with BM-MSCs had decreased tau hyperphosphorylation and improved cognitive function. In conclusion, BM-MSCs can modulate immune/inflammatory responses in AD mice, ameliorate their pathophysiology, and improve the cognitive decline associated with Abeta deposits. These results demonstrate that BM-MSCs are a potential new therapeutic agent for AD.

Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells.

Abstract: The generation of induced pluripotent stem cells (iPSC) has enormous potential for the development of patient-specific regenerative medicine Human embryonic stem cells (hESC) are able to defend their genomic integrity by maintaining low levels of reactive oxygen species (ROS) through a combination of enhanced removal capacity and limited production of these molecules Such limited ROS production stems partly from the small number of mitochondria present in hESC; thus, it was important to determine that human iPSC (hiPSC) generation is able to eliminate the extra mitochondria present in the parental fibroblasts (reminiscent of "bottleneck" situation after fertilization) and to show that hiPSC have antioxidant defenses similar to hESC We were able to generate seven hiPSC lines from adult human dermal fibroblasts and have fully characterized two of those clones Both hiPSC clones express pluripotency markers and are able to differentiate in vitro into cells belonging to all three germ layers One of these clones is able to produce fully differentiated teratoma, whereas the other hiPSC clone is unable to silence the viral expression of OCT4 and c-MYC, produce fully differentiated teratoma, and unable to downregulate the expression of some of the pluripotency genes during the differentiation process In spite of these differences, both clones show ROS stress defense mechanisms and mitochondrial biogenesis similar to hESC Together our data suggest that, during the reprogramming process, certain cellular mechanisms are in place to ensure that hiPSC are provided with the same defense mechanisms against accumulation of ROS as the hESC

Subventricular Zone-Derived Neural Progenitor Cells Migrate Along a Blood Vessel Scaffold Toward the Post-stroke Striatum

Abstract: The subventricular zone (SVZ) of the adult brain contains neural stem cells that have the capacity to regenerate new neurons after various insults. Brain ischemia causes damage to brain tissue and induces neural regeneration together with angiogenesis. We previously reported that, after ischemic injury in mice, SVZ-derived neural progenitor cells (NPCs) migrate into the striatum, and these NPCs are frequently associated with blood vessels in the regenerating brain tissue. Here we studied the role of blood vessels during the neural regeneration in more detail. BrdU administration experiments revealed that newly generated NPCs were associated with both newly formed and pre-existing blood vessels in the ischemic striatum, suggesting that the angiogenic environment is not essential for the neuron-blood vessel interaction. To observe migrating NPCs and blood vessels simultaneously in damaged brain tissue, we performed live imaging of cultured brain slices after ischemic injury. In this system, we virally labeled SVZ-derived NPCs in Flk1-EGFP knock-in mice in which the blood vessels are labeled with EGFP. Our results provide direct evidence that SVZ-derived NPCs migrate along blood vessels from the SVZ toward the ischemic region of the striatum. The leading process of the migrating NPCs was closely associated with blood vessels, suggesting that this interaction provides directional guidance to the NPCs. These findings suggest that blood vessels play an important role as a scaffold for NPCs migration toward the damaged brain region.

Concise Review: Bone Marrow-Derived Stem/Progenitor Cells in Cutaneous Repair and Regeneration

Abstract: Our understanding of the role of bone marrow (BM)-derived cells in cutaneous homeostasis and wound healing had long been limited to the contribution of inflammatory cells. Recent studies, however, suggest that the BM contributes a significant proportion of noninflammatory cells to the skin, which are present primarily in the dermis in fibroblast-like morphology and in the epidermis in a keratinocyte phenotype; and the number of these BM-derived cells increases markedly after wounding. More recently, several studies indicate that mesenchymal stem cells derived from the BM could significantly impact wound healing in diabetic and nondiabetic animals, through cell differentiation and the release of paracrine factors, implying a profound therapeutic potential. This review discusses the most recent understanding of the contribution of BM-derived noninflammatory cells to cutaneous homeostasis and wound healing.

Epithelial‐Mesenchymal Transition‐Derived Cells Exhibit Multilineage Differentiation Potential Similar to Mesenchymal Stem Cells

Abstract: The epithelial-to-mesenchymal transition (EMT) is an embryonic process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows breast epithelial cells with stem cell traits. In this report, we have further characterized the EMT-derived cells and shown that these cells are similar to mesenchymal stem cells (MSCs) with the capacity to differentiate into multiple tissue lineages. For this purpose, we induced EMT by ectopic expression of Twist, Snail, or transforming growth factor-beta in immortalized human mammary epithelial cells. We found that the EMT-derived cells and MSCs share many properties including the antigenic profile typical of MSCs, that is, CD44(+), CD24(-), and CD45(-). Conversely, MSCs express EMT-associated genes, such as Twist, Snail, and mesenchyme forkhead 1 (FOXC2). Interestingly, CD140b (platelet-derived growth factor receptor-beta), a marker for naive MSCs, is exclusively expressed in EMT-derived cells and not in their epithelial counterparts. Moreover, functional analyses revealed that EMT-derived cells but not the control cells can differentiate into alizarin red S-positive mature osteoblasts, oil red O-positive adipocytes and alcian blue-positive chondrocytes similar to MSCs. We also observed that EMT-derived cells but not the control cells invade and migrate towards MDA-MB-231 breast cancer cells similar to MSCs. In vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSCs. In conclusion, we have demonstrated that the EMT-derived cells are similar to MSCs in gene expression, multilineage differentiation, and ability to migrate towards tumor cells and wound sites.

Cardiospheres Recapitulate a Niche-Like Microenvironment Rich in Stemness and Cell-Matrix Interactions, Rationalizing Their Enhanced Functional Potency for Myocardial Repair†‡§

Abstract: Cardiac stem cells (CSCs) are promising candidates for use in myocardial regenerative therapy. We test the hypothesis that growing cardiac-derived cells as three-dimensional cardiospheres may recapitulate a stem cell niche-like microenvironment, favoring cell survival and enhancing functional benefit after transplantation into the injured heart. CSCs and supporting cells from human endomyocardial biopsies were grown as cardiospheres and compared with cells cultured under traditional monolayer condition or dissociated from cardiospheres. Cardiospheres self-assembled into stem cell niche-like structures in vitro in suspension culture, while exhibiting greater proportions of c-kit(+) cells and upregulated expression of SOX2 and Nanog. Pathway-focused polymerase chain reaction (PCR) array, quantitative real-time PCR, and immunostaining revealed enhanced expression of stem cell-relevant factors and adhesion/extracellular-matrix molecules (ECM) in cardiospheres including IGF-1, histone deacetylase 2 (HDAC2), Tert, integrin-α(2), laminin-β(1), and matrix metalloproteinases (MMPs). Implantation of cardiospheres in severe combined immunodeficiency (SCID) mouse hearts with acute infarction disproportionately improved cell engraftment and myocardial function, relative to monolayer-cultured cells. Dissociation of cardiospheres into single cells decreased the expression of ECM and adhesion molecules and undermined resistance to oxidative stress, negating the improved cell engraftment and functional benefit in vivo. Growth of cardiac-derived cells as cardiospheres mimics stem cell niche properties with enhanced "stemness" and expression of ECM and adhesion molecules. These changes underlie an increase in cell survival and more potent augmentation of global function following implantation into the infarcted heart.