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Oligodendrocytes (OLs) are responsible for myelin production and metabolic support of neurons. Defects in OLs are crucial in several neurodegenerative diseases including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). This protocol describes a method to generate oligodendrocyte precursor cells (OPCs) from human pluripotent stem cells (hPSCs) in only ~20 d, which can subsequently myelinate neurons, both in vitro and in vivo. To date, OPCs have been derived from eight different hPSC lines including those derived from patients with spontaneous and familial forms of MS and ALS, respectively. hPSCs, fated for 8 d toward neural progenitors, are transduced with an inducible lentiviral vector encoding for SOX10. The addition of doxycycline for 10 d results in >60% of cells being O4-expressing OPCs, of which 20% co-express the mature OL marker myelin basic protein (MBP). The protocol also describes an alternative for viral transduction, by incorporating an inducible SOX10 in the safe harbor locus AAVS1, yielding ~100% pure OPCs. O4+ OPCs can be purified and either cryopreserved or used for functional studies. As an example of the type of functional study for which the derived cells could be used, O4+ cells can be co-cultured with maturing hPSC-derived neurons in 96/384-well-format plates, allowing the screening of pro-myelinating compounds.

Inflammation has been recognized as essential for restorative regeneration. Here, we analyzed the sequential processes during onset of liver injury and subsequent regeneration based on time-resolved transcriptional regulatory networks (TRNs) to understand the relationship between inflammation, mature organ function, and regeneration. Genome-wide expression and TRN analysis were performed time dependently in mouse liver after acute injury by CCl4 (2 h, 8 h, 1, 2, 4, 6, 8, 16 days), as well as lipopolysaccharide (LPS, 24 h) and compared to publicly available data after tunicamycin exposure (mouse, 6 h), hepatocellular carcinoma (HCC, mouse), and human chronic liver disease (non-alcoholic fatty liver, HBV infection and HCC). Spatiotemporal investigation differentiated lobular zones for signaling and transcription factor expression. Acute CCl4 intoxication induced expression of gene clusters enriched for inflammation and stress signaling that peaked between 2 and 24 h, accompanied by a decrease of mature liver functions, particularly metabolic genes. Metabolism decreased not only in pericentral hepatocytes that underwent CCl4-induced necrosis, but extended to the surviving periportal hepatocytes. Proliferation and tissue restorative TRNs occurred only later reaching a maximum at 48 h. The same upstream regulators (e.g. inhibited RXR function) were implicated in increased inflammation and suppressed metabolism. The concomitant inflammation/metabolism TRN occurred similarly after acute LPS and tunicamycin challenges, in chronic mouse models and also in human liver diseases. Downregulation of metabolic genes occurs concomitantly to induce inflammation-associated genes as an early response and appears to be initiated by similar upstream regulators in acute and chronic liver diseases in humans and mice. In the acute setting, proliferation and restorative regeneration associated TRNs peak only later when metabolism is already suppressed. ispartof: ARCHIVES OF TOXICOLOGY vol:94 issue:1 pages:205-217 ispartof: location:Germany status: published

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications., Mechanical forces, along with gene regulatory networks and cell-cell signalling, play an important role in the complex organization of tissues. Here the authors describe devices that actively apply mechanical force to developing neural tube, demonstrating that mechanical forces increase growth and enhance patterning.

Lymphatic capillary growth is an integral part of wound healing, yet, the combined effectiveness of stem/progenitor cells on lymphatic and blood vascular regeneration in wounds needs further exploration. Stem/progenitor cell transplantation also emerged as an approach to cure lymphedema, a condition caused by lymphatic system deficiency. While lymphedema treatment requires lymphatic system restoration from the capillary to the collector level, it remains undetermined whether stem/progenitor cells support a complex regenerative response across the entire anatomical spectrum of the system. Here, we demonstrate that, although multipotent adult progenitor cells (MAPCs) showed potential to differentiate down the lymphatic endothelial lineage, they mainly trophically supported lymphatic endothelial cell behaviour in vitro. In vivo, MAPC transplantation supported blood vessel and lymphatic capillary growth in wounds and restored lymph drainage across skin flaps by stimulating capillary and pre-collector vessel regeneration. Finally, human MAPCs mediated survival and functional reconnection of transplanted lymph nodes to the host lymphatic network by improving their (lymph)vascular supply and restoring collector vessels. Thus, MAPC transplantation represents a promising remedy for lymphatic system restoration at different anatomical levels and hence an appealing treatment for lymphedema. Furthermore, its combined efficacy on lymphatic and blood vascular growth is an important asset for wound healing.

Peripheral vascular disease (PVD) is a growing medical problem in Western societies and presents itself mainly in two different clinical forms. Intermittent claudication is an early moderate manifestation, while patients with critical limb ischemia suffer from severe muscle tissue loss or ulcers and are at high risk for limb amputation. Unfortunately, many patients cannot be helped with currently available surgical or endovascular revascularization procedures because of the complex anatomy of the vascular occlusion and/or the presence of other risk factors. Noninvasive stem cell therapy has been proposed as an alternative for such patients. Although pioneering clinical experience with stem cell-related therapy seems promising, it is too early for general clinical use of this technique, since many questions remain unanswered. Indeed, while questions about safety, dose, and administration route/timing/frequency are the first ones to be addressed when designing a stem cell-based clinical approach, there is accumulating evidence from recent (pre-)clinical studies that other issues may also be at stake. For instance, the choice of stem cells to be used and its precise mechanism of action, the need/possibility for concurrent tissue regeneration in case of irreversible tissue loss, the differentiation degree and specific vascular identity of the transplanted cells, and the long-term survival of engrafted cells in the absence of a normal supportive tissue environment should be well considered. Here, rather than presenting a comprehensive and extensive overview on the current literature on stem/progenitor cells and revascularization, we highlight some of the outstanding issues emerging from the recent (pre-)clinical literature that may codetermine the successful application of stem cells in a wide range of PVD patients in the future. ispartof: Journal of Molecular Medicine vol:87 issue:1 pages:3-16 ispartof: location:Germany status: published

The Notch signaling pathway is a multifunctional, evolutionarily conserved pathway, which plays an important role in development as well as stem cell biology. Multipotent adult progenitor cells (MAPCs) represent a unique stem cell population, which is capable of differentiating into cell types of the ectodermal, mesodermal and endodermal lineages in vitro, and contribute to most somatic cell types in vivo. Our aim was to characterize the gene expression of Notch signaling elements in rodent MAPCs. We show that transcripts for Notch-receptors, ligands, regulatory molecules of the pathway and the Hairy/Enhancer of Split-1 (HES-1) target gene are present in mouse and rat low-Oct4 MAPCs. We found that mouse Notch3 and rat Notch1 transcripts increased when cells were cultured at high density for 48 to 96 hours. HES-1 and HES-related transcription factor-1 (HERP-1), transcriptional targets of Notch-signaling, were both elicited by immobilized Delta1 ligand. In addition, mRNA for Notch1 and Notch3 was also induced by Notch-signaling, suggesting the presence of regulatory feedback loops. Slight differences between mouse and rat derived MAPCs suggest that the exact function, transcriptional regulation and the fine-tuning of the signal may be species specific. Taken together, we characterized the gene expression profile of the Notch pathway in rodent low-Oct4-MAPCs, and showed that the pathway is functional and can be modulated. Our results provide an additional tool and a further basis for a better understanding of stem cell biology. ispartof: Pathology Oncology Research vol:13 issue:4 pages:302-310 ispartof: location:Switzerland status: published

Immune mechanisms have been shown to contribute to the process of myelodysplastic syndromes (MDS)-related bone marrow (BM) failure. The aim of this study was to evaluate the possible contribution of activated monocytes through CD40-CD40L(CD154) interactions with activated T helper cells. We demonstrated in 77 predominantly lower risk MDS patients that the CD40 receptor was expressed significantly higher on monocytes and that CD40L was expressed significantly higher on T helper cells in peripheral blood (PB) and BM. Increased levels of CD40 and CD40L were detected in the same patients. In addition, stimulation of the CD40 receptor on purified PB monocytes led to a significantly higher tumor necrosis factor alpha production in patients. Co-culture of BM mononuclear cells of 21 patients in the presence of a blocking CD40 monoclonal antibody (ch5D12) led to a significant increase in the number of colony-forming units. A correlation was seen between increased CD40 expression on monocytes with patients' age below 60 years and with the cytogenetic abnormality trisomy 8. These results demonstrate that CD40 expression on monocytes may identify a subgroup of MDS patients in whom immune-mediated hematopoietic failure is part of the disease process. As such, the CD40-CD40L-based activation of monocytes might be a target to counteract MDS-related BM failure. ispartof: Leukemia vol:21 issue:12 pages:2411-2419 ispartof: location:England status: published

Recent registry studies have established umbilical cord blood (UCB) transplantation as a safe and feasible alternative to bone marrow transplantation in adults when no sibling donor is available. There is, however, no gold standard to guide optimal treatment choices. We review here factors leading to the choice of the 'best available donor' and 'best available unit' in the case of UCB. For instance, it is clear that higher cell dose may partially overcome the negative impact of certain histocompatibility leukocyte antigen (HLA) disparities in UCB transplantation, leading us to choose the more closely HLA-matched unit with a cell dose >2.5 x 10(7)/kg. New approaches in adult UCB transplantation are systematically covered, with a quantitative appreciation of the evidence available to date. Reduced intensity conditioning, for example, broadens the range of potential recipients by reducing transplant-related mortality, but suffers from unproven risks and benefits long term. Potential advantages of multiple units over single unit transplants are discussed, with a particular emphasis on confounding factors that impact interpretation. The limited clinical results of ex vivo UCB expansion, the possible benefits of co-infusion of haploidentical cells and controversial issues (e.g. killer immunoglobulin-like receptor matching and alternative graft sources) are also addressed with a debate on the future of UCB transplantation. ispartof: Bone Marrow Transplantation vol:38 issue:2 pages:83-93 ispartof: location:England status: published

Stem cells are defined by their biological function. A stem cell is an undifferentiated cell that self-renews to maintain the stem cell pool and at the single-cell level differentiates into more than one mature, functional cell. In addition, when transplanted, a stem cell should be capable of replacing a damaged organ or tissue for the lifetime of the recipient. Some would argue that stem cells should also be capable of functionally integrating into nondamaged tissues. Stem cells are critical to both embryogenesis and postnatal life. ispartof: Stem Cell Reviews vol:1 issue:1 pages:53-59 ispartof: location:United States status: published

The progenitor content of autologous peripheral blood progenitor and stem cell collections is a major determinant of prompt hematopoietic recovery following autologous stem cell transplantation. We analyzed unstimulated bone marrow (BM) and peripheral blood (PB) apheresis products in comparison to those collected following G-CSF or GM-CSF stimulation. We quantitated their committed (CFU-GM) and primitive (long-term culture-initiating cells, LTC-IC) progenitors in relation to hematologic recovery in 63 patients undergoing autografting for lymphoid malignancies. G-CSF, but not GM-CSF, substantially enriched the committed progenitor content (2.5-3.6-fold) of both PB and BM grafts. G-CSF also enriched the LTC-IC content of BM and PB compared to control grafts. GM-CSF augmented (11.5-fold) the LTC-IC content of stimulated BM, but not GM-CSF-mobilized PB. Neutrophil recovery was substantially quicker in recipients of BM or PB mobilized with G-CSF or GM-CSF. In contrast, red cell and platelet recovery was accelerated in recipients of GM-CSF-stimulated BM (but not PB) and G-CSF-stimulated PB (but not BM). No direct correlation between progenitor dose and hematopoietic recovery for neutrophils, platelets or red cells was observed. Cytokine stimulation can augment the committed and more primitive multilineage progenitor content of BM and PB grafts, to a differing extent. The uncertain relationship with multilineage myeloid recovery emphasizes the limitations in using clonogenic progenitor analyses to assess the adequacy of an autologous graft prior to transplantation. ispartof: Bone marrow transplantation vol:32 issue:6 pages:575-80 ispartof: location:England status: published

BCR/ABL: from molecular mechanisms of leukemia induction to treatment of chronic myelogenous leukemia

Chronic myelogenous leukemia (CML), characterized by the BCR-ABL gene rearrangement, has been extensively studied. Significant progress has been made in the area of BCR-ABL-mediated intracellular signaling, which has led to a better understanding of BCR-ABL-mediated clinical features in chronic phase CML. Disease progression and blast crisis CML is associated with characteristic non-random cytogenetic and molecular events. These can be viewed as increased oncogenic activity or loss of tumor suppressor activity. However, what causes transformation and disease progression to blast crisis is only poorly understood. This is in part due to the lack of a good in vivo model of chronic phase CML even though animal models developed over the last few years have started to provide insights into blast crisis development. Thus, additional in vitro and in vivo studies will be needed to provide a complete understanding of the contribution of BCR-ABL and other genes to disease progression and to improve therapeutic approaches for blast crisis CML. ispartof: Leukemia vol:16 issue:8 pages:1402-11 ispartof: location:England status: published

Multiple sources of HSCs exist. Here, Verfaillie discusses the long-term engraftment capabilities of each source and the search for ex vivo expansion conditions to allow bulk culture for therapeutic HSC transplantion. ispartof: Nature immunology vol:3 issue:4 pages:314-7 ispartof: location:United States status: published

Abnormal beta1 integrin receptor function may contribute to the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors in chronic myelogenous leukemia (CML). Previous studies suggest that abnormal integrin function in CML progenitors is related to the presence of the BCR/ABL oncogene. BCR/ABL may alter integrin function in CML by phosphorylating cytoskeletal and/or signaling proteins important for normal integrin function. We evaluated the effect of Tyrphostin AG957, a protein tyrosine kinase (PTK) inhibitor which has activity against the p210BCR/ABL kinase, on beta1 integrin function in CML progenitors. Incubation of CML marrow CD34+HLA-DR+ cells with Tyrphostin AG957 at concentrations that did not affect colony-forming cells (CFC) viability, but which partly inhibited p210BCR/ABL kinase activity, significantly increased CML CFC adhesion to stroma and alpha4beta1 and alpha5beta1 integrin binding fragments of fibronectin (FN). CML CFC proliferation, unlike that of normal CFC, is not inhibited following integrin receptor engagement with FN or anti-integrin antibodies. AG957 did not alter CML CFC proliferation by itself, but resulted in significant inhibition of CML CFC proliferation following integrin engagement. Another PTK inhibitor, Tyrphostin AG555, which does not have anti-p210BCR/ABL kinase activity, did not affect CML CFC adhesion or proliferation. Neither AG957 nor AG555 affected normal CFC adhesion or proliferation. In BCR/ABL expressing cells, AG957 partially inhibited phosphorylation of several proteins that are BCR/ABL PTK substrates and are involved in normal integrin signaling. These studies suggest that abnormal tyrosine phosphorylation may play an important role in defective integrin function in CML progenitors.

Adhesion of normal colony-forming cells (CFC) to bone marrow (BM) stroma and the extracellular matrix (ECM) component fibronectin (FN) depends at least in part on the alpha4beta1 and alpha5beta1 integrins and the CD44 receptor. Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal CFC is associated with inhibition of their proliferation. In contrast to normal CFC, chronic myelogenous leukemia (CML) Ph+ CFC adhere significantly less to either stroma or FN. CML Ph+ CFC proliferation is also not inhibited by coculture with stroma or FN. However, equal numbers of alpha4, alpha5, and beta1 integrins and CD44 are present on CML and normal CD34+ cells. We have previously demonstrated that beta1-dependent adhesion to and subsequent proliferation inhibition by FN can be restored when CML Ph+ CFC are incubated with the beta1 integrin activating antibody, 8A2, and demonstrated a role for the alpha5beta1 integrin in this phenomenon. Since the integrin alpha4beta1 and the proteoglycan form of CD44 may cooperate in establishing normal CFC adhesion to FN, we examined if treatment of CML Ph+ CFC with 8A2 also restores the cooperativity between beta1 integrins and CD44. We demonstrate that 8A2 induces adhesion of CML Ph+ CFC not only to intact FN but also to alpha4beta1, alpha5beta1, and proteoglycan binding fragments of FN. 8A2-induced adhesion to these fragments and peptides also results in a significant inhibition of the proliferation of CML Ph+ CFC. Addition of antibodies to either the alpha5, alpha4, or beta1 integrins, antibodies against the CD44 receptor, or removal of chondroitin sulfate glycosaminoglycans from the surface of CML CD34+ HLA-DR+ cells significantly reduced the 8A2-induced adhesion to and adhesion-mediated inhibition of proliferation by FN. These studies demonstrate that activation of beta1 integrins on CML Ph+ CFC not only results in upregulation of beta1 integrin-dependent adhesion and adhesion-mediated inhibition of proliferation, but also in the restoration of cooperation between beta1 integrins and CD44. These studies suggest that decreased beta1 integrin avidity may also affect the function of the proteoglycan adhesion receptor CD44, both of which may contribute to the abnormal circulation and expansion of malignant progenitors in CML.

CML, characterized by the BCR/ABL gene rearrangement has been more extensively studied than any other malignancy. Over the last decade, significant progress has been made in our understanding of BCR-ABL-induced alterations in intracellular signaling. Unfortunately, we still only poorly understand the correlation between the clinical symptoms of chronic phase CML and the BCR-ABL oncoprotein. This is in part due to lack of a good in vivo animal model of chronic phase CML. In vivo and in vitro studies from the Clarkson group, recently reviewed in this journal (Leukemia 1997; 11: 1404-1428), have significantly enhanced our understanding of the pathophysiology of CML. However, further characterization of the effect of the BCR-ABL oncoprotein on signal molecules involved with cell differentiation, cell proliferation, cell survival and cell adhesion in primary Ph+ CML progenitors or in vivo models of CML will be needed to provide a full understanding of the pathophysiology of chronic phase CML.

Nature 418, 41–49 (2002); doi:10.1038/nature00870; published online 20 June 2002 In response to questions raised recently about flow cytometry data reported in Fig. 1 of this Article, we had the flow cytometry data for Fig. 1 and the experimental approach used to generate them reviewed by flow cytometry experts.

Comparison of the transcriptomes of pluripotent embryonic stem cells, multipotent adult progenitor cells and lineage restricted mesenchymal stem cells identified a unique gene expression profile of multipotent adult progenitor cells., Background Recently, several populations of postnatal stem cells, such as multipotent adult progenitor cells (MAPCs), have been described that have broader differentiation ability than classical adult stem cells. Here we compare the transcriptome of pluripotent embryonic stem cells (ESCs), MAPCs, and lineage-restricted mesenchymal stem cells (MSCs) to determine their relationship. Results Applying principal component analysis, non-negative matrix factorization and k-means clustering algorithms to the gene-expression data, we identified a unique gene-expression profile for MAPCs. Apart from the ESC-specific transcription factor Oct4 and other ESC transcripts, some of them associated with maintaining ESC pluripotency, MAPCs also express transcripts characteristic of early endoderm and mesoderm. MAPCs do not, however, express Nanog or Sox2, two other key transcription factors involved in maintaining ESC properties. This unique molecular signature was seen irrespective of the microarray platform used and was very similar for both mouse and rat MAPCs. As MSC-like cells isolated under MAPC conditions are virtually identical to MSCs, and MSCs cultured in MAPC conditions do not upregulate MAPC-expressed transcripts, the MAPC signature is cell-type specific and not merely the result of differing culture conditions. Conclusion Multivariate analysis techniques clustered stem cells on the basis of their expressed gene profile, and the genes determining this clustering reflected the stem cells' differentiation potential in vitro. This comparative transcriptome analysis should significantly aid the isolation and culture of MAPCs and MAPC-like cells, and form the basis for studies to gain insights into genes that confer on these cells their greater developmental potency.