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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.

MG53 is an important membrane repair protein and partially protects bone marrow multipotent adult progenitor cells (MAPCs) against oxidized low-density lipoprotein (ox-LDL). The present study was to test the hypothesis that the limited protective effect of MG53 on MAPCs was due to ox-LDL-induced reduction of MG53. MAPCs were cultured with and without ox-LDL (0-20 μg/mL) for up to 48 hours with or without MG53 and antioxidant N-acetylcysteine (NAC). Serum MG53 level was measured in ox-LDL-treated mice with or without NAC treatment. Ox-LDL induced significant membrane damage and substantially impaired MAPC survival with selective inhibition of Akt phosphorylation. NAC treatment effectively prevented ox-LDL-induced reduction of Akt phosphorylation without protecting MAPCs against ox-LDL. While having no effect on Akt phosphorylation, MG53 significantly decreased ox-LDL-induced membrane damage and partially improved the survival, proliferation and apoptosis of MAPCs in vitro. Ox-LDL significantly decreased MG53 level in vitro and serum MG53 level in vivo without changing MG53 clearance. NAC treatment prevented ox-LDL-induced MG53 reduction both in vitro and in vivo. Combined NAC and MG53 treatment significantly improved MAPC survival against ox-LDL. These data suggested that NAC enhanced the protective effect of MG53 on MAPCs against ox-LDL through preventing ox-LDL-induced reduction of MG53. ispartof: JOURNAL OF CELLULAR AND MOLECULAR MEDICINE vol:24 issue:1 pages:886-898 ispartof: location:England status: published

Hematopoietic stem/progenitor cells (HSPCs) are responsible for the generation of blood cells throughout life. It is believed that, in addition to soluble cytokines and niche cells, biophysical cues like elasticity and oxygen tension are responsible for the orchestration of stem cell fate. Although several studies have examined the effects of bone marrow (BM) niche elasticity on HSPC behavior, no study has yet investigated the effects of the elasticity of other niche sites like the fetal liver (FL), where HSPCs expand more extensively. In this study, we evaluated the effect of matrix stiffness values similar to those of the FL on BM-derived HSPC expansion. We first characterized the elastic modulus of murine FL tissue at embryonic day E14.5. Fibrin hydrogels with similar stiffness values as the FL (soft hydrogels) were compared with stiffer fibrin hydrogels (hard hydrogels) and with suspension culture. We evaluated the expansion of total nucleated cells (TNCs), Lin&minus, /cKit+ cells, HSPCs (Lin&minus, /Sca+/cKit+ (LSK) cells), and hematopoietic stem cells (HSCs: LSK- Signaling Lymphocyte Activated Molecule (LSK-SLAM) cells) when cultured in 5% O2 (hypoxia) or in normoxia. After 10 days, there was a significant expansion of TNCs and LSK cells in all culture conditions at both levels of oxygen tension. LSK cells expanded more in suspension culture than in both fibrin hydrogels, whereas TNCs expanded more in suspension culture and in soft hydrogels than in hard hydrogels, particularly in normoxia. The number of LSK-SLAM cells was maintained in suspension culture and in the soft hydrogels but not in the hard hydrogels. Our results indicate that both suspension culture and fibrin hydrogels allow for the expansion of HSPCs and more differentiated progeny whereas stiff environments may compromise LSK-SLAM cell expansion. This suggests that further research using softer hydrogels with stiffness values closer to the FL niche is warranted.

Hematopoietic stem cell (HSC) maintenance throughout life and engraftment after transplantation during anticancer treatment, depend on support from various bone marrow (BM) niche compartments. Here, by studying physiological homing of fetal HSCs, we show the critical requirement of balanced crosstalk within the multi-component BM niche for enabling HSC-lodgment. Transgene-induced over-production of VEGF by skeletal progenitor cells elicited stromal and endothelial hyper-activation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSC seeding, settlement and survival were drastically impaired. Transcriptome-profiling, flow-cytometry and high-resolution imaging indicated alterations in peri-/endothelial characteristics, vascular permeability, and cellular metabolism, associated with a higher oxidative status and increased oxidative stress within the VEGF-enriched BM-environment. Thus, developmental HSC-homing to bone is controlled by local inter-niche communication regulating stromal-vascular integrity and theoxidative and metabolic milieu.Since irradiation of adult mice also induced stromal VEGF-expression and similar osteo-angiogenic niche changes, our findings can contribute to improving stem cell therapy.

Postnatal hematopoietic stem (and progenitor) cells (HS(P)Cs) are especially vulnerable to oxidative stress, leading to early hematopoietic senescence and/or malignant transformation. Elevated intracellular reactive oxygen species (ROS) can, among others, oxidize nucleotides, and thus can result in genotoxicity and mutagenesis if left unrepaired. Oxidized bases, as well as other spontaneous single base modifications, are recognized and repaired by the base excision repair (BER) pathway. Hence, the BER pathway is crucial to maintain genome integrity. In contrast to other DNA repair pathways however, the role of BER in maintaining HSPC functionality remains enigmatic, chiefly because knockout (KO) of BER genes is in many cases embryonic lethal. BER is a complex multi-step repair process. After initial removal and excision of the damaged base, the apurinic/apyrimidinic (AP) site is processed by the AP endonuclease (APEX1) enzyme. At this point, the BER pathway branches into 2 sub-pathways, namely the short-patch (SP-BER; wherein DNA polymerase beta (Polβ), Ligase III (Lig3) together with X-ray repair cross-complementing protein 1 (Xrcc1) are active) and the long-patch BER (LP-BER; wherein Lig1, Flap Structure-Specific Endonuclease 1 (Fen1), and sometimes Polβ are active) for the repair synthesis and the gap filling steps. In this study we wished to address the role of BER in adult hematopoiesis. Therefore, we used CRISPR-Cas9 to KO different BER genes in adult bone marrow (BM) HS(P)Cs, including two genes common to the BER (sub-)pathway(s) (Apex1 and Polβ) as well as one gene in the SP-BER (Xrcc1) and one gene in the LP-BER (Lig1) pathway. The effect thereof was evaluated on HS(P)C repopulation in vivo as well as on HS(P)C expansion during long-term in vitro culture (using the culture medium described by Wilkinson et al., Nature 2019). All CRISPR-Cas9 experiments were validated using a second sgRNA targeting the selected BER genes. Lig1-KO caused in vivo HSPC dysfunction: at 20 weeks post-transplantation, significantly less Lig1 KO cells were observed in the committed progenitor (HPC) and lineage committed (Lin +) BM compartments. By contrast, KO of Xrcc1 had only minor effects on HS(P)C repopulation, but we observed increased HSC expansion and myeloid biased differentiation in some recipient mice, which might correspond to clonal hematopoiesis and is consistent with the finding of XRCC1 loss-of-function mutation in myelodysplastic patients (Joshi et al, Ann Hematol 2016). Knockout of Polβ did not affect hematopoiesis in vivo or in vitro. The most severe phenotype was observed when we knocked out Apex1, as Apex1-KO HS(P)Cs failed to repopulate irradiated recipient mice. Already after 2 weeks, significantly less Apex1 deficient cells were detected in the different blood lineages and nearly no CRISPR-Cas9 KO cells could be detected from 4 weeks onwards. This was confirmed in vitro, where reduced expansion of Apex1 KO BM cells was observed. APEX1 has two major functional activities, namely its nuclease activity, involved in BER, and its redox activity (also called Ref-1 function) important in reducing oxidized transcription factors and therefore implicated in transcriptional regulation. However, little is known regarding the nuclease and Ref-1 function(s) in primary adult hematopoietic cells. We therefore cultured BM HS(P)Cs for 1 week in the continuous presence of 2 distinct chemicals blocking the APEX1 nucleases, or 2 different chemicals inhibiting specifically the Ref-1 function. We demonstrated that both APEX1 functions are essential for hematopoiesis, even if the 2 functions appear to support the survival, expansion and maintenance of HS(P)Cs through different mechanisms. While the Ref-1 function was essential for proliferation (as both Ref-1 inhibitors cause cell cycle arrest) of all the lineages (including the Lin + cells), both inhibitors of the nuclease function affected more the expansion/survival of the less committed HS(P)Cs without leading to any cell cycle arrest. In conclusion, this study demonstrates for the first time the important role of BER genes in adult hematopoiesis, often deregulated in cancer, including hematopoietic malignancies. We observed a particularly severe phenotype upon loss of Apex1 in adult HSPCs, and ongoing studies (such as RNA sequencing analysis) should provide novel insights in underlying mechanisms of APEX1 deficiencies in HS(P)Cs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8+ cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was—even after major histocompatibility complex class I upregulation—insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8−CD69+ T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Significance Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy and have the advantage over mesenchymal stem cells (MSCs) of large-scale manufacturing and banking potential and thus prompt availability, it is important to understand how MAPCs interact with immune cells to validate their widespread therapeutic applicability. Cytotoxic immune effector cells play a crucial role in immune homeostasis and in the pathogenesis of some autoimmune diseases. This study assessed for the first time the in vitro influence of a clinical-grade human MAPC product (MultiStem) on the cytotoxic function of CD8+ T cells (CTLs) by evaluating the immunogenicity of MAPCs and the susceptibility of MAPCs toward CTL-mediated lysis and by analyzing the mechanism of MAPC-mediated modulation of CTL functionality. These results may represent a highly relevant contribution to the current knowledge and, in combination with the results of future phase II/III trials using MultiStem, could lead to an intriguing continuation of stem cell-based research for immunotherapy.

Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14 transcription factors (TFs) in reprogramming MAPCs to induced endodermal progenitor cells (iENDO cells), defined as cells that can be long-term expanded and differentiated to both hepatocyte- and endocrine pancreatic-like cells. We demonstrated that 14 TF-iENDO cells can be expanded for at least 20 passages, differentiate spontaneously to hepatocyte-, endocrine pancreatic-, gut tube-like cells as well as endodermal tumor formation when grafted in immunodeficient mice. Furthermore, iENDO cells can be differentiated in vitro into hepatocyte- and endocrine pancreatic-like cells. However, the pluripotency TF OCT4, which is not silenced in iENDO cells, may contribute to the incomplete differentiation to mature cells in vitro and to endodermal tumor formation in vivo. Nevertheless, the studies presented here provide evidence that reprogramming of adult stem cells to an endodermal intermediate progenitor, which can be expanded and differentiate to multiple endodermal cell types, might be a valid alternative for the use of PSCs for creation of endodermal cell types. ispartof: PLOS ONE vol:13 issue:5 ispartof: location:United States status: published

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.

Pluripotent stem cells have the property of long-term self-renewal and the potential to give rise to descendants of the three germ layers and hence all mature cells in the human body. Therefore, they hold the promise of offering insight not only into human development but also for human disease modeling and regenerative medicine. However, the generation of mature differentiated cells that closely resemble theircounterparts remains challenging. Recent advances in single-cell transcriptomics and computational modeling of gene regulatory networks are revealing a better understanding of lineage commitment and are driving modern genome editing approaches. Additional modification of the chemical microenvironment, as well as the use of bioengineering tools to recreate the cellular, extracellular matrix, and physical characteristics of the niche wherein progenitors and mature cells reside, is now being used to further improve the maturation and functionality of stem cell progeny. ispartof: F1000Research vol:7 pages:220- ispartof: location:England status: published

© 2018 Joke Terryn et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background and Aims: Human pluripotent stem cell- (PSC-) derived neurons are increasingly used in the study of neurodevelopmental and neurodegenerative diseases. Electrophysiological activity is a crucial and defining function of these neurons. Patch clamping and the more recently developed multielectrode arrays are used to evaluate this complex function. In this study, we assess the effect of topography on PSC-derived neurons as a baseline for the development of next-generation silicon oxide substrates (in direct relation to MEA fabrication) that integrate topography as a means to guide network formation and stimulate differentiation. Methods: Human PSC was differentiated towards cortical neurons and seeded on a silicon micropatterned substrate to screen for topographic guidance and improved neurite outgrowth. We next developed customized micropillar and microgroove substrates to further assess the effect of topography on human cortical neurons. Immunocytochemistry and morphological analyses were used to track neuron differentiation and guidance. Results: Microgroove and micropillar substrates appeared to support the differentiation of human stem cell-derived cortical neurons, promoted neurite outgrowth, and facilitated contact guidance. In contrast to flat surfaces, topography also appeared to limit the number of NESTIN positive neuronal progenitor cells present in the culture. Conclusion: We describe the topographical preferences of human PSC-derived cortical neurons, on neurite outgrowth and guidance. Embedding topography in silicon substrates may be an effective approach to direct and improve neuronal network formation. We propose to integrate topography in multielectrode arrays as a practical method to shape the neuro-electronic interface. ispartof: JOURNAL OF NANOMATERIALS vol:2018 status: published

Background aims Myelodysplastic syndromes (MDS) are a group of clonal stem cell disorders affecting the normal hematopoietic differentiation process and leading to abnormal maturation and differentiation of all blood cell lineages. Treatment options are limited, and there is an unmet medical need for effective therapies for patients with severe cytopenias. Methods We demonstrate that multipotent adult progenitor cells (MAPC) improve the function of hematopoietic progenitors derived from human MDS bone marrow (BM) by significantly increasing the frequency of primitive progenitors as well as the number of myeloid colonies. Results This effect was more pronounced in a non-contact culture, indicating the importance of soluble factors produced by the MAPC cells. Moreover, the cells did not stimulate the growth of the abnormal MDS clone, as shown by fluorescent in situ hybridization analysis on BM cells from patients with a known genetic abnormality. We also demonstrate that MAPC cells can provide stromal support for patient-derived hematopoietic cells. When MAPC cells were intravenously injected into a mouse model of MDS, they migrated to the site of injury and increased the hematopoietic function in diseased mice. Discussion The preclinical studies undertaken here indicate an initial proof of concept for the use of MAPC cell therapy in patients with MDS-related severe and symptomatic cytopenias and should pave the way for further investigation in clinical trials.

Background: Primary graft dysfunction (PGD) is considered to be the end result of an inflammatory response targeting the new lung allograft after transplant. Previous research has indicated that MAPC cell therapy might attenuate this injury by its paracrine effects on the pro-/anti-inflammatory balance. This study aims to investigate the immunoregulatory capacities of MAPC cells in PGD when administered in the airways. Methods: Lungs of domestic pigs (n = 6/group) were subjected to 90 minutes of warm ischemia. Lungs were cold flushed, cannulated on ice and placed on EVLP for 6 hours. At the start of EVLP, 40 ml of an albumin-plasmalyte mixture was distributed in the airways (CONTR group). In the MAPC cell group, 150 million MAPC cells (ReGenesys/Athersys, Cleveland, OH, USA) were added to this mixture. At the end of EVLP, a physiological evaluation (pulmonary vascular resistance, lung compliance, PaO2/FiO(2)), wet-to-dry weight ratio (W/D) sampling and a multiplex analysis of bronchoalveolar lavage (BAL) (2 x 30 ml) was performed. Results: Pulmonary vascular resistance, lung compliance, PaO2/FiO(2) and W/D were not statistically different at the end of EVLP between both groups. BAL neutrophilia was significantly reduced in the MAPC cell group. Moreover, there was a significant decrease in TNF-alpha, IL-1 beta and IFN-gamma in the BAL, but not in IFN-alpha; whereas IL-4, IL-10 and IL-8 were below the detection limit. Conclusions: Although no physiologic effect of MAPC cell distribution in the airways was detected during EVLP, we observed a reduction in pro-inflammatory cytokines and neutrophils in BAL in the MAPC cell group. This effect on the innate immune system might play an important role in critically modifying the process of PGD after transplantation. Further experiments will have to elucidate the immunoregulatory effect of MAPC cell administration on graft function after transplantation.

Aims The present study was to investigate the effect of hydrogen peroxide (H2O2) on bone marrow stem cells and their endothelial differentiation and the underlying mechanisms in vitro. Main methods Rat bone marrow multipotent adult progenitor cells (MAPCs) were used as the source of bone marrow stem cells, and treated with H2O2 (with the final concentration from 0 to 50 μM) with or without N-acetylcysteine (NAC, 0.1 mM). Reactive oxygen species (ROS) was measured by electron paramagnetic resonance (EPR) and fluorescent microscope. Flow cytometry and immunoblotting were used to determine apoptosis and differentiation of MAPCs. Key findings H2O2 generated a significant amount of intracellular and extracellular ROS in the culture system, substantially inhibited the proliferation of MAPCs and Oct-4 expression, and induced their apoptosis in a dose-dependent manner. Exposure to H2O2 also significantly attenuated the endothelial differentiation of MAPCs with reduced expression of endothelial markers CD31 and FLK-1 as well as impaired in vitro vascular structure formation. Both intracellular and extracellular ROS production from H2O2 were blocked by NAC. NAC treatment effectively prevented H2O2-induced reduction of Oct-4 expression in the cells. However, NAC treatment only partially prevented H2O2-induced apoptosis, and inhibition of cell proliferation and endothelial differentiation of MAPCs. Significance H2O2 exposure suppressed Oct-4 expression in MAPCs through ROS-dependent mechanism, while increasing the apoptosis of MAPCs and inhibiting their proliferation and endothelial differentiation with a mechanism partially due to ROS generation in vitro.

Background Potent immunomodulatory effects have been reported for mesenchymal stem/stromal cells (MSCs), multipotent adult progenitor cells (MAPCs), and fibroblasts. However, side-by-side comparisons of these cells specifically regarding immunophenotype, gene expression, and suppression of proliferation of CD4+ and CD8+ lymphocyte populations have not been reported. Methods We developed MAPC and MSC lines from rhesus macaque bone marrow and fibroblast cell lines from rhesus dermis and assessed phenotypes based upon differentiation potential, flow cytometric analysis of immunophenotype, and quantitative RT-PCR analysis of gene expression. Using allogeneic lymphocyte proliferation assays, we compared the in vitro immunomodulatory potency of each cell type. Results and Conclusions Extensive phenotypic similarities exist among each cell type, although immunosuppressive potencies are distinct. MAPCs are most potent, and fibroblasts are the least potent cell type. All three cell types demonstrated immunomodulatory capacity such that each may have potential therapeutic applications such as in organ transplantation, where reduced local immune response is desirable.

Directional migration determines hematopoietic stem/progenitor cell (HSPC) homing, which depends upon the interaction between the chemokine CXCL12 and its receptor CXCR4. CD26 is a widely expressed membrane-bound ectopeptidase that cleaves CXCL12 thereby depleting its chemokine activity. We identified tissue-factor pathway inhibitor (TFPI) as a biological inhibitor of CD26 in murine and human HSPCs. We observed low-level TFPI expression in endothelial cells in the bone marrow (BM), which did not increase following radiation injury. Treatment of HSPCs with TFPI in vitro led to enhanced HSPC migration toward CXCL12, as well as homing and engraftment in the BM upon transplantation. We found that Glypican-3 (GPC3), a heparan sulfate proteoglycan expressed on murine as well as human HSPCs, mediated this effect. TFPI did not affect CD26 activity, migration, or homing of GPC3(-/-) HSPCs, while it affected GPC1(-/-) HSPCs similar to wild-type HSPCs. Moreover, proliferation of GPC3(-/-) but not GPC1(-/-) BM HSPCs was significantly increased, which was associated with a decrease in the primitive HSC pool in BM and an increase in proportion of the circulating HSPCs in the peripheral blood. Hence, we present a novel role for TFPI and GPC3 in regulating HSC homing as well as retention in the BM.

This article discusses the implications of the recent discovery that rat bone marrow-derived multipotent adult progenitor cells (rMAPCs), a cell type with broad somatic differentiation potential but of uncertain lineage identity, are similar to rat blastocyst-derived extraembryonic endoderm precursor (rXENP) cells, which appear to represent the committed extraembryonic endoderm precursor of the blastocyst. It was found that under rMAPC culture conditions, rXENP cells can be homogeneously cultured and similar cells, named rat hypoblast stem cells (rHypoSCs), can be derived from rat blastocysts more rapidly and directly. The detailed comparison of rHypoSCs, rXENP cells, and rMAPCs revealed highly similar gene expression profiles and developmental potentials. The significance of these findings for embryology, stem cell biology, and medicine is discussed. Specifically, the results assign a lineage identity to rMAPCs, indicate that rMAPCs originated by environmental reprogramming, and imply that HypoSCs, XENP cells, and MAPCs possess lineage plasticity. The MAPC-HypoSC relation also strengthens the consistency of rat and mouse embryology and consequently the idea that HypoSCs represent the committed extraembryonic endoderm precursor of the blastocyst. On this basis, it is argued that the direct comparison of HypoSCs (now available in pure form) with embryonic stem cells will be highly useful for the understanding of pluripotency and plasticity. Finally, the new findings suggest an explanation for an obscure observation on stem cell-induced transplantation tolerance. Thus, the HypoSC/XENP/MAPC phenotype provides a unique but broadly instructive model with which to study stem cell plasticity, reprogramming, and transplantation tolerance, all central themes in regenerative medicine. STEM CELLS2013;31:620–626

Because of their extended differentiation capacity, stem cells have gained great interest in the field of regenerative medicine. For the development of therapeutic strategies, more knowledge on the in vivo fate of these cells has to be acquired. Therefore, stem cells can be labeled with radioactive tracer molecules such as 18F-FDG, a positron-emitting glucose analog that is taken up and metabolically trapped by the cells. The aim of this study was to optimize the radioactive labeling of mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) in vitro with 18F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM). Methods: Mouse MSCs and rat MAPCs were used for 18F-FDG uptake kinetics and tracer retention studies. Cell metabolic activity, proliferation, differentiation and ultrastructural changes after labeling were evaluated using an Alamar Blue reagent, doubling time calculations and quantitative TEM, respectively. Additionally, mice were injected with MSCs and MAPCs prelabeled with 18F-FDG, and stem cell biodistribution was investigated using small-animal PET. Results: The optimal incubation period for 18F-FDG uptake was 60 min. Significant early tracer washout was observed, with approximately 30%–40% of the tracer being retained inside the cells 3 h after labeling. Cell viability, proliferation, and differentiation capacity were not severely affected by 18F-FDG labeling. No major changes at the ultrastructural level, considering mitochondrial length, lysosome size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed with TEM. Small-animal PET experiments with radiolabeled MAPCs and MSCs injected intravenously in mice showed a predominant accumulation in the lungs and a substantial elution of 18F-FDG from the cells. Conclusion: MSCs and MAPCs can be successfully labeled with 18F-FDG for molecular imaging purposes. The main cellular properties are not rigorously affected. TEM confirmed that the cells’ ultrastructural properties are not influenced by 18F-FDG labeling. Small-animal PET studies confirmed the intracellular location of the tracer and the possibility of imaging injected prelabeled stem cell types in vivo. Therefore, direct labeling of MSCs and MAPCs with 18F-FDG is a suitable technique to noninvasively assess cell delivery and early retention with PET.

Somatic, also termed adult, stem cells are highly attractive biomedical cell candidates because of their extensive replication potential and functional multilineage differentiation capacity. They can be used for drug and toxicity screenings in preclinical studies, as in vitro model to study differentiation or for regenerative medicine to aid in the repair of tissues or replace tissues that are lost upon disease, injury or ageing. Multipotent adult progenitor cells (MAPCs) and mesenchymal stem cells (MSCs) are two types of adult stem cells derived from bone marrow that are currently being used clinically for tissue regeneration and for their immunomodulatory and trophic effects. This review will give an overview of the phenotypic and functional differences between human MAPCs and MSCs, with a strong emphasis on their immunological characteristics. Finally, we will discuss the clinical studies in which MSCs and MAPCs are already used.

Objectives: Membrane repair mechanism is important to the cell survival and function. MG53, also known as TRIM72, is a critical component in the membrane repair process. We have demonstrated that cell membrane damage was involved in the impaired survival of bone marrow stem cells (BMSCs) by oxidized low-density lipoprotein (ox-LDL) in vitro, and recombinant human MG53 protein (rhMG53) significantly protected the cell membrane of BMSCs and enhanced their survival. However, the mechanism(s) for ox-LDL-induced membrane damage in BMSCs is unclear. It is well known that ox-LDL is a potent oxidative agent with formation of abundant reactive oxygen species ( ROS ). The present study was designed to determine the role of ROS in ox-LDL-induced membrane damage in BMSCs. Methods: Rat bone marrow multipotent adult progenitor cells ( MAPCs ) were cultured with and without ox-LDL (0-20 ug/ml) for up to 48 hrs. Exposure of MAPCs to ox-LDL led to increased LDH release to the media and entry of fluorescent dye FM1-43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox-LDL also generated excessive ROS as measured with electron paramagnetic resonance spectroscopy. While antioxidant N-acetylcysteine (NAC) completely blocked ROS production from ox-LDL, it failed to prevent ox-LDL-induced cell death or membrane damage. When MAPCs were treated with rhMG53, ox-LDL induced LDH release and FM1-43 dye entry were significantly reduced. Interestingly, NAC treatment significantly potentiated the protective effect of rhMG53 on ox-LDL-induced membrane damage with enhanced cell survival. Western blot analysis showed that ox-LDL significantly reduced the level of rhMG53 both in vitro and in vivo that was substantially prevented with NAC treatment. Conclusion: Our data suggest that membrane damage induced by ox-LDL contributed to the impaired survival of MAPCs independent of ROS production. NAC potentiated the protective effect of rhMG53 on ox-LDL-induced membrane damage of BMSCs via preventing ox-LDL-induced reduction of rhMG53 level.

Multipotent adult progenitor cells (MAPCs) are isolated from bone marrow-derived mesenchymal stem cell cultures. Human MAPCs (commercially available as a clinical-grade product termed MultiStem®) possess the capacity to be expanded long term; however, the differentiation potential of human MAPC is restricted mainly to the mesodermal lineage. Both rodent and human MAPCs have been shown to be effective in improving outcome of preclinical models of ischemic cardiac, vascular and neurological diseases, where the mechanism of action is mainly via trophic, cytoprotective, and/or immunomodulatory effects, as actual cellular replacement is rare. Human MAPCs are currently being evaluated in a number of phase I/II clinical trials in patients with stroke, acute myocardial infarction (AMI), inflammatory bowel disease (IBD), and in patients undergoing BM transplantation for the prevention of acute graft-versus-host disease (GvHD).

Tumor infiltrating stem cells have been suggested as a vehicle for the delivery of a suicide gene towards otherwise difficult to treat tumors like glioma. We have used herpes simplex virus thymidine kinase expressing human multipotent adult progenitor cells in two brain tumor models (hU87 and Hs683) in immune-compromised mice. In order to determine the best time point for the administration of the codrug ganciclovir, the stem cell distribution and viability were monitored in vivo using bioluminescence (BLI) and magnetic resonance imaging (MRI). Treatment was assessed by in vivo BLI and MRI of the tumors. We were able to show that suicide gene therapy using HSV-tk expressing stem cells can be followed in vivo by MRI and BLI. This has the advantage that (1) outliers can be detected earlier, (2) GCV treatment can be initiated based on stem cell distribution rather than on empirical time points, and (3) a more thorough follow-up can be provided prior to and after treatment of these animals. In contrast to rodent stem cell and tumor models, treatment success was limited in our model using human cell lines. This was most likely due to the lack of immune components in the immune-compromised rodents. The authors thank Mrs. Ann Van Santvoort for technical assistance with the acquisition of in vivo images. They thank the Leuven Viral Vector Core for the construction and production of viral vectors used in this study. Funding was provided by the Flemish government for the IWT SBO iMAGiNe (080017) and NanoCoMIT (140061), the European Commission for FP7-NMP-2008-228933 ("Vibrant"), and the University of Leuven for PF IMIR (10/017).

UNLABELLED: The role of progenitor cells in liver repair and fibrosis has been extensively described, but their purification remains a challenge, hampering their characterization and use in regenerative medicine. To address this issue, we developed an easy and reproducible liver progenitor cell (LPC) isolation strategy based on aldehyde dehydrogenase (ALDH) activity, a common feature shared by many progenitor cells. We demonstrate that a subset of nonparenchymal mouse liver cells displays high levels of ALDH activity, allowing the isolation of these cells by fluorescence-activated cell sorting. Immunocytochemistry and qPCR analyses on freshly isolated ALDH(+) cells reveal an enrichment in cells expressing liver stem cell markers such as EpCAM, CK19, CD133, and Sox9. In culture, the ALDH(+) population can give rise to functional hepatocyte-like cells as illustrated by albumin and urea secretion and cytochrome P450 activity. ALDH1A1 expression can be detected in canals of Hering and bile duct epithelial cells and is increased on liver injury. Finally, we showed that the isolation and differentiation toward hepatocyte-like cells of LPCs with high ALDH activity is also successfully applicable to human liver samples. CONCLUSION: High ALDH activity is a feature of LPCs that can be taken advantage of to isolate these cells from untreated mouse as well as human liver tissues. This novel protocol is practically relevant, because it provides an easy and nontoxic method to isolate liver stem cells from normal tissue for potential therapeutic purposes.

Conditions such as muscular dystrophies (MDs) that affect both cardiac and skeletal muscles would benefit from therapeutic strategies that enable regeneration of both of these striated muscle types. Protocols have been developed to promote induced pluripotent stem cells (iPSCs) to differentiate toward cardiac or skeletal muscle; however, there are currently no strategies to simultaneously target both muscle types. Tissues exhibit specific epigenetic alterations; therefore, source-related lineage biases have the potential to improve iPSC-driven multilineage differentiation. Here, we determined that differential myogenic propensity influences the commitment of isogenic iPSCs and a specifically isolated pool of mesodermal iPSC-derived progenitors (MiPs) toward the striated muscle lineages. Differential myogenic propensity did not influence pluripotency, but did selectively enhance chimerism of MiP-derived tissue in both fetal and adult skeletal muscle. When injected into dystrophic mice, MiPs engrafted and repaired both skeletal and cardiac muscle, reducing functional defects. Similarly, engraftment into dystrophic mice of canine MiPs from dystrophic dogs that had undergone TALEN-mediated correction of the MD-associated mutation also resulted in functional striatal muscle regeneration. Moreover, human MiPs exhibited the same capacity for the dual differentiation observed in murine and canine MiPs. The findings of this study suggest that MiPs should be further explored for combined therapy of cardiac and skeletal muscles. ispartof: Journal of Clinical Investigation vol:125 issue:12 pages:4463-4482 ispartof: location:United States status: published

Hepatocyte-like cells derived from stem cells hold great potential for clinical and pharmaceutical applications, including high-throughput drug toxicity screening. We report a three-dimensional aggregate culture system for the directed differentiation of adult rat bone marrow-derived stem cells, rat multipotent adult progenitor cells, to hepatocyte-like cells. Compared to adherent monolayer cultures, differentiation in the aggregate culture system resulted in significantly higher expression level of liver-specific transcripts, including an increased albumin mRNA level, and higher levels of albumin and urea secretion. This coincides with the presence of significantly more cells that express intracellular albumin at levels found in primary hepatocytes. The differentiated cell aggregates exhibited cytochrome P450-mediated ethoxyresorufin-O-dealkylation and pentoxyresorufin-O-dealkylation activity. Consistent with these increased mature functions, cells within the aggregates were shown to have many ultrastructural features of mature hepatocytes by transmission electron microscopy. With the scalability of the aggregate culture system and the enhanced differentiation capability, this system may facilitate translation of generating hepatocytes from stem cells to technology.

Multipotent adult progenitor cells (MAPC) are clinically being explored as an alternative to mesenchymal stem cells (MSC) for the immunomodulatory control of graft-versus-host disease (GvHD). Here, we performed an explorative study of the immunomodulatory potential of mouse MAPC (mMAPC), in comparison with that of MSC (mMSC) using experimental models of T-cell alloreactivity. Suppressive effects of Oct4-expressing mMAPC have been described previously; here, we studied mMAPC expressing low to no Oct4 ('mClone-3'), recently shown to be most representative for the human MAPC counterpart. mClone-3 and mMSC exhibited similar immunophenotype and in vitro immunogenic behavior. Allogeneic T-cell↔dendritic cell-proliferation assays showed strong dose-dependent T-cell-suppressive effects of both mClone-3 and mMSC. In a popliteal lymph node assay, mClone-3 and mMSC equally suppressed in vivo alloreactive T-cell expansion. We conclude that mouse MAPC and MSC exhibit similar immunosuppressive behavior in in vitro and local in vivo GvHD assays.

Mesoangioblasts (MABs) are a subset of muscle-derived pericytes able to restore dystrophic phenotype in mice and dogs. However, their lifespan is limited and they undergo senescence after 25-30 population doublings. Recently, induced pluripotent stem cells (iPSCs) generated from reprogrammed fibroblasts have been demonstrated to have in vitro and in vivo myogenic potential when sorted for the SM/C-2.6 antigen. Furthermore, chimeric mice from mdx-iPSCs (DYS-HAC) cells showed tissue-specific expression of dystrophin. Nevertheless, myogenic differentiation protocols and the potential of iPSCs generated from different cell sources still present unanswered questions. Here we show that iPSCs generated from prospectively sorted MABs (MAB-iPSCs) are pluripotent as fibroblast-derived iPSCs (f-iPSCs). However, both teratoma formation and genetic cell manipulation assays identify a durable epigenetic memory in MAB-iPSCs, resulting in stronger myogenic commitment. Striated muscle tissue accounts for up to 70% of MAB-iPSC teratomas. Moreover, transfection with Pax3 and Pax7 induces a more robust myogenic differentiation in MAB-iPSCs than in f-iPSCs. A larger amount of CD56(+) progenitors can be sorted from the MAB-iPSCs differentiating pool and, after transplantation into αsg-KO mice, can efficiently participate to skeletal muscle regeneration and restore αsg expression. Our data strongly suggest that iPSCs are a heterogeneous population and, when generated from myogenic adult stem cells, they exhibit a stronger commitment, paving the way for creating custom-made cell protocols for muscular dystrophies.

This study was to investigate the effect of oxidized low-density lipoprotein (ox-LDL) on the behaviour of bone marrow stem cells and their endothelial differentiation as well as the underlying mechanisms. Adult rat bone marrow multipotent progenitor cells (MAPCs) were incubated with ox-LDL for up to 2 weeks. Ox-LDL treatment resulted in a time- and dose-dependent reduction of MAPC population in culture through a combination of decreased cell proliferation and increased apoptosis. The expression of stem cell marker Oct-4 was significantly suppressed in MAPCs by ox-LDL in a dose- and time-dependant manner. Endothelial differentiation of MAPCs was substantially inhibited by ox-LDL with markedly decreased expression of endothelial markers vWF, Flk-1 and CD31, as well as impaired in vitro vascular structure formation. Ox-LDL-induced apoptosis and inhibition of Oct-4 expression, cell proliferation and endothelial differentiation of MAPCs were associated with significant inhibition of Akt phosphorylation. Akt overexpression in MAPCs transfected with a constitutively active Akt completely reversed the effects of ox-LDL on MAPCs including enhanced apoptosis, decreased cell proliferation, suppressed Oct-4 expression and endothelial differentiation as well as in vitro vascular structure formation. In conclusion, ox-LDL promotes apoptosis and inhibits Oct-4 expression and self-renewal of MAPCs, and impairs their endothelial differentiation via suppression of Akt signalling.

Non-viral integrating systems, PhiC31 phage integrase (ϕC31), andSleeping Beautytransposase (SB), provide an effective method forex vivogene delivery into cells. Here, we used a plasmid-encoding GFP and neomycin phosphotransferase along with recognition sequences for bothϕC31 and SB integrating systems to demonstrate that both systems effectively mediated integration in cultured human fibroblasts and in rat multipotent adult progenitor cells (rMAPC). Southern blot analysis of G418-resistant rMAPC clones showed a 2-fold higher number of SB-mediated insertions per clone compared toϕC31. Sequence identification of chromosomal junction sites indicated a random profile for SB-mediated integrants and a more restricted profile forϕC31 integrants. Transgenic rMAPC generated with both systems maintained their ability to differentiate into liver and endothelium albeit with marked attenuation of GFP expression. We conclude that both SB andϕC31 are effective non-viral integrating systems for genetic engineering of MAPC in basic studies of stem cell biology.

Proteinases have been implicated in the mobilization of haematopoietic progenitor cells (HPCs) from the bone marrow (BM). Here, we report the involvement of the plasminogen (Plg) system in the haematopoietic recovery following chemotherapy. By using gene-deficient mice, we found that plasmin and its activators tPA and uPA play a role in the haematopoietic recovery upon delivery of the cytotoxic agent 5-fluoro-uracil (5-FU). The impaired haematopoietic recovery of Plg-deficient (Plg(-/-)) mice after 5-FU was not rescued by depletion of fibrinogen, indicating that it was not due to defective fibrinolysis. Instead, loss of Plg impaired breakdown of fibronectin, VCAM-1 and laminin-BM matrix proteins involved in adhesion of HPCs to their BM microenvironment and in transendothelial migration of HPCs. These findings provide novel insights in how plasmin regulates haematopoietic recovery upon cytotoxic myeloablation.

Sphere-forming cochlear stem cells/progenitors have recently been isolated from the postnatal mouse organ of Corti. However, self-renewal of the cochlear progenitors remains to be elucidated. Here we demonstrate the renewable cochlear neural progenitors from the postnatal Immortomouse organ of Corti containing a temperature-sensitive SV40 that have the potential to differentiate into hair cell-, neuron-, and supporting cell-like phenotypes under the guidance of sonic hedgehog, epidermal growth factor, retinoic acid, and brain-derived neurotrophic factor , herein termed SERB . This work provides a rationale for using cochlear neural progenitors in possible cell replacement of lost hair cells and neurons in degenerative hearing disorders.

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

PURPOSE OF REVIEW Cardiovascular diseases are the leading cause of morbidity/mortality in the world. Despite significant progress in cardiovascular medicine, mortality rates have remained steady during the past decade. Therefore, investigators are evaluating novel methods to repair/regenerate the damaged heart. RECENT FINDINGS Within the past decade, stem cell therapy strategies have been evaluated for the treatment of cardiovascular diseases. Among all postnatal stem cell compartments, bone marrow has been most extensively studied. Recent studies using bone marrow stem cells have suggested that this approach may have beneficial effects on cardiac function, probably by enhancing new cell formation from endogenous cardiac and endothelial stem/progenitor cells. SUMMARY The present review critically assesses the possible beneficial effects of bone-marrow-derived cell therapy in cardiac ischemia, demonstrating benefits through as yet incompletely understood mechanisms.

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

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40-80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP(+)CD45.2(+) cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP(+) MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 10(3)-fold more MAPCs were required for efficient engraftment. Because GFP(+) host-derived CD45.1(+) cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs. ispartof: Journal of Experimental Medicine vol:204 issue:1 pages:129-139 ispartof: location:United States status: published

Rationale: Recent studies described the association between hematopoietic stem/progenitor cell (HSPC) expansion in the bone marrow (BM), leukocytosis in the peripheral blood and accelerated atherosclerosis. Objective: We hypothesized that circulating HSPC may home to inflamed vessels, where they might contribute to inflammation and neointima formation. Methods and results: We demonstrated that Lin- Sca-1(+) cKit(+) (LSK cells) in bone marrow (BM) and peripheral blood of LDLr(-/-) mice on high fat diet expressed significantly more integrin β2, which was responsible for LSK cell adhesion and migration toward ICAM-1 in vitro, and homing to injured arteries in vivo, all of which were blocked with an anti-CD18 blocking antibody. When homed LSK cells were isolated from ligated artery and injected to irradiated recipients, they resulted in BM reconstitution. Injection of CD18(+/+) LSK cells to immunodeficient Balb/C Rag2- Ɣ C(-/-) recipients resulted in more severe inflammation and reinforced neointima formation in the ligated carotid artery, compared to mice injected with PBS and CD18(-/-) LSK cells. Hypercholesterolemia stimulated ERK phosphorylation (pERK) in LSK cells of LDLr(-/-) mice in vivo. Blockade of pERK reduced ARF1 expression, leading to decreased integrin β2 function on HSPC. In addition, integrin β2 function could be regulated via ERK-independent LRP1 pathway. Conclusions: Integrin β2 expression on HSPC is regulated by hypercholesterolemia, specifically LDL, in pERK dependent and independent manners, leading to increased homing and localization of HSPC to injured arteries, which is highly correlated with arteriosclerosis. This article is protected by copyright. All rights reserved. ispartof: Stem Cells vol:4 issue:4 pages:1230-1240 ispartof: location:England status: published

Multipotent adult progenitor cells (MAPCs) are marrow-derived pluripotent stem cells with a broad differentiation potential. We sought to identify factors that affect adoptively transferred MAPCs. In vitro, MAPCs expressed low levels of major histocompatibility complex (MHC) antigens, failed to stimulate CD4(+) and CD8(+) T-cell alloresponses, and were targets of NK cytolysis. To study in vivo biodistribution, we labeled MAPCs with luciferase for sequential quantification of bioluminescence and DsRed2 for immunohistochemical analysis. C57BL /6 MAPCs were infused intravenously into C57BL /6, Rag-2(-/-) (T- and B-cell-deficient), and Rag-2(-/-)/IL-2Rgamma(c)(-/-) (T-, B-, and NK-cell-deficient) mice. In C57BL /6 mice, MAPCs were transiently detected only in the chest compared with long-term persistence in T- and B-cell-deficient mice. NK depletion reduced MAPC elimination. Because the lungs were the major uptake site after intravenous injection, intra-arterial injections were tested and found to result in more widespread biodistribution. Widespread MAPC biodistribution and long-term persistence were seen in irradiated recipients given allogeneic marrow and MAPCs; such MAPCs expressed MHC class I antigens in tissues. Our data indicate that the biodistribution and persistence of reporter gene-labeled MAPCs are maximized after intra-arterial delivery or host irradiation and that T cells, B cells, and NK cells contribute to in vivo MAPC rejection. ispartof: Blood vol:107 issue:10 pages:4182-4188 ispartof: location:United States status: published

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus, reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly, however, PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process, PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1, the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. ispartof: Journal of Clinical Investigation vol:116 issue:5 pages:1230-1242 ispartof: location:United States status: published

Cell therapy with bone marrow stem cells (BMSCs) remains a viable option for tissue repair and regeneration. A major challenge for cell therapy is the limited cell survival after implantation. This study was to investigate the effect of oxidized low-density lipoprotein (ox-LDL, naturally present in human blood) on BMSC injury and the effect of MG53, a tissue repair protein, for the improvement of stem cell survival. Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL, which caused significant cell death as reflected by the increased LDH release to the media. Exposure of MAPCs to ox-LDL led to entry of fluorescent dye FM1-43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox-LDL also generated reactive oxygen species (ROS) as measured with electron paramagnetic resonance spectroscopy. While antioxidant N-acetylcysteine completely blocked ROS production from ox-LDL, it failed to prevent ox-LDL-induced cell death. When MAPCs were treated with the recombinant human MG53 protein (rhMG53) ox-LDL induced LDH release and FM1-43 dye entry were significantly reduced. In the presence of rhMG53, the MAPCs showed enhanced cell survival and proliferation. Our data suggest that membrane damage induced by ox-LDL contributed to the impaired survival of MAPCs. rhMG53 treatment protected MAPCs against membrane damage and enhanced their survival which might represent a novel means for improving efficacy for stem cell-based therapy for treatment of diseases, especially in setting of hyperlipidemia. ispartof: Journal of Cellular and Molecular Medicine vol:18 issue:12 pages:2445-53 ispartof: location:England status: published

Stem cells have the capacity for self renewal and undergo multilineage differentiation. Stem cells isolated from both blastocysts and adult tissues represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self‐renewal and differentiation capacity. In generating particular cell types for specific applications, it is important to direct their differentiation to the desired lineage. In vitro differentiation of stem cells usually produces a mixed population of different cell lineages with the desired cell type present only at a small proportion. Use of growth factors that promote differentiation, and expansion or survival of specific cell types are key in controlling the differentiation towards specific cell lineages. Our limited knowledge of their growth conditions as well as lack of appropriate markers associated with different stages of differentiation hinders the widespread ...

Previous studies have identified Sleeping Beauty transposons as efficient vectors for nonviral gene delivery in mammalian cells. However, studies demonstrating the usefulness of transposons as gene delivery vehicles into adult stem cells are lacking. Multipotent adult progenitor cells (MAPC) are nonhematopoietic stem cells with the capacity to form most, if not all, cell types of the body and as such hold great therapeutic potential. The whole-body biodistribution and persistence of MAPC are unknown, and such data would help direct clinical applications. We have nucleofected murine MAPC with two plasmid-based Sleeping Beauty transposons encoding the red fluorescent protein (DsRed2) and firefly luciferase. Transgenic euploid MAPC clones maintained their characteristic multilineage differentiation potential in vitro. DsRed2 and luciferase expression allowed for MAPC detection in vivo and in tissue sections. To confirm that transgenesis occurred by transposition into the genome of MAPC, we mapped Sleeping Beauty transposon integration sites in two MAPC clones using splinkerette PCR. This novel dual-reporter imaging approach based on the transgenesis of MAPC with Sleeping Beauty transposons sheds light on the homing patterns of MAPC and paves the way for quantification of MAPC engraftment in real time in vivo. ispartof: Molecular Therapy vol:12 issue:1 pages:42-48 ispartof: location:United States status: published

OBJECTIVE: We have previously shown that hematopoietic progenitor cells (HPCs) from umbilical cord blood (UCB) can be maintained in a cytokine-supplemented stroma-noncontact (SNC) system. Here, we tested if all-trans retinoic acid (ATRA), known to improve expansion of murine hematopoietic stem cells, would enhance human HPC maintenance in a SNC culture system. METHODS: CD34+CD38-Lin- cells from UCB were cultured in transwells above AFT024 in the presence of Flt-3 ligand (FLT) and thrombopoietin (TPO), with or without ATRA. Total nucleated cells (TNC), colony-forming units (CFUs), long-term culture-initiating cells (LTC-ICs), myeloid-lymphoid initiating cells (ML-ICs) and SCID repopulating cells (SRCs) were evaluated 1 to 5 weeks after culture. RESULTS: All-trans retinoic acid (1 mumol/L) reduced expansion of CD34+CD38-Lin- TNC and CFUs after 2 to 5 weeks of culture. However, it significantly increased LTC-IC expansion after 1 to 3 and, even more so, 5 weeks of culture. ATRA also increased recovery of more primitive ML-ICs and SRCs. Increased HPC recovery appeared dependent on the presence of stromal cells, as LTC-IC expansion was significantly reduced when ATRA was added to stroma-free cultures. CONCLUSION: All-trans retinoic acid increases expansion of early HPCs in a stromal cell-dependent fashion. ispartof: Experimental Hematology vol:33 issue:4 pages:422-427 ispartof: location:Netherlands status: published

We investigated the mechanisms of intermediate cell mass (ICM) expansion in zebrafish chordin (Chd) morphant embryos and examined the role of BMPs in relation to this phenotype. At 24 h post-fertilization (hpf), the expanded ICM of embryos injected with chd morpholino (MO) (ChdMO embryos) contained a monotonous population of hematopoietic progenitors. In situ hybridization showed that hematopoietic transcription factors were ubiquitously expressed in the ICM whereas vascular gene expression was confined to the periphery. BMP4 (but not BMP2b or 7) and smad5 mRNA were ectopically expressed in the ChdMO ICM. At 48 hpf, monocytic cells were evident in both the ICM and circulation of ChdMO but not WT embryos. While injection of BMP4 MO had no effect on WT hematopoiesis, co-injecting BMP4 with chd MOs significantly reduced ICM expansion. Microarray studies revealed a number of genes that were differentially expressed in ChdMO and WT embryos and their roles in hematopoiesis has yet to be determined. In conclusion, the expanded ICM in ChdMO embryos represented an expansion of embryonic hematopoiesis that was skewed towards a monocytic lineage. BMP4, but not BMP2b or 7, was involved in this process. The results provide ground for further research into the mechanisms of embryonic hematopoietic cell expansion. ispartof: Developmental Biology vol:277 issue:1 pages:235-254 ispartof: location:United States 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

Multipotent adult progenitor cells (MAPC) are stem cells isolated primarily from adult bone marrow that have the ability to differentiate in vitro into cells with phenotypic and functional characteristics of cells from the three germ layers, namely endoderm, mesoderm and neuroectoderm. In addition, MAPCs proliferate for extended periods of time without obvious senescence. Hence, MAPCs may be ideal cells for therapy of genetic disorders, provided that the main impediment to gene therapy, namely efficient gene transfer and persistent gene expression, can be overcome. Most commercially available lipid-based methods that very highly efficiently transfect cell lines and primary cells, fail to transfect MAPC. However, 10–15% transfection of MAPC can be achieved using Superfect. However, this approach requires high density culture of MAPCs which subsequently differentiate. Between 12 and 15% transfection is achieved in MAPCs using electroporation, but this is highly toxic to MAPCs. Here, using transient expression of an enhanced green fluorescent protein (EGFP) gene, we report that nucleofection results in a transfection efficiency of over 25% with mouse MAPC without perturbing the conditions suitable for MAPC growth and maintenance, and with significantly less toxicity than electroporation. Similar results were seen for rat and human MAPC. Efficient transfection of MAPC by nucleofection offers an appealing non-viral mode of gene delivery and can be used to overexpress genes of interest in these cells. In addition, it should easily accomodate emerging site-specific integration technology, thereby culminating eventually in MAPC-mediated long-term gene therapy for genetic disorders.

OBJECTIVE: Hematopoietic progenitor proliferation and differentiation are inhibited by integrin engagement of fibronectin (FN). Focal adhesion kinases have been shown to mediate intracellular signaling from integrins, and we recently demonstrated that gene expression and pre-mRNA splicing of the focal adhesion kinase, PYK2, is abnormal in CD34(+) cells from chronic myelogenous leukemia (CML) patients. Here we investigated whether PYK2 gene products mediate integrin signaling in hematopoietic stem and progenitor cells. METHODS: Cord blood CD34(+) cells were retrovirally transduced with vectors encoding Pyk2H, Pyk2, or the dominant negative-acting, kinase-deficient, C-terminal PYK2 fragment, PRNK, and myeloid proliferation and differentiation was assessed using colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and liquid culture assays. RESULTS: CD34(+) cells overexpressing Pyk2H or Pyk2 generated 50% less colony-forming unit granulocyte/macrophage (CFU-GM) than eGFP-transduced controls. Although the number of CFC generated by PRNK-expressing cells was unchanged, LTC-IC were significantly reduced. Culture of CD34(+) cells on FN significantly reduced the generation of mature myeloid cells vs those cultured on BSA-coated wells, and could be overcome by addition of SCF. As is observed when integrins are engaged, overexpression of either Pyk2H or Pyk2 decreased committed myeloid progenitor proliferation and differentiation; however, SCF could not override this inhibition. Finally, as is observed when integrins are not engaged, PRNK-mediated inhibition of endogenous Pyk2H resulted in integrin-nonresponsive proliferation and differentiation of myeloid precursors and accelerated differentiation of primitive hematopoietic progenitors. CONCLUSION: These studies indicate that PYK2 gene products mediate integrin-induced signals that regulate myelopoiesis. ispartof: Experimental hematology vol:32 issue:4 pages:365-74 ispartof: location:Netherlands status: published

We recently showed that a rare cell from murine bone marrow, which we termed multipotent adult progenitor cells (MAPCs), can be expanded for >120 population doublings. Mouse (m)MAPCs differentiate into mesenchymal lineage cells as well as endothelium and endoderm, and, when injected in the blastocyst, mMAPCs contribute to most if not all somatic cell lineages including the different cell types of the brain. Our results, reported herein, demonstrate that mMAPCs can also be induced to differentiate into cells having anatomical and electrophysiological characteristics similar to those of midbrain neurons. Differentiation to a neuronal phenotype was achieved by coculturing mMAPCs with astrocytes, suggesting that neuronal differentiation may require astrocyte-derived factors similar to what is required for the differentiation of embryonic stem cells and neural stem cells to neurons. Differentiation of mMAPCs to neuron-like cells follows similar developmental steps as described for embryonic stem cells and neural stem cells. MAPCs therefore may constitute a source of cells for treatment of central nervous system disorders. ispartof: pages:11854-60 ispartof: Proceedings of the National Academy of Sciences of the United States of America vol:100 issue:Suppl 1 pages:11854-60 ispartof: Arthur M Sackler Colloquium of the National-Academy-of-Sciences on Regenerative Medicine location:CA, IRVINE date:18 Oct - 22 Oct 2002 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

We have identified a population of primitive cells in normal human (h) as well as murine (m) and rat (r), postnatal bone marrow (BM) that have, at the single cell level, multipotent differentiation and extensive proliferation potential, which we named Multipotent Adult Progenitor Cell or MAPC. MAPC differentiate in vitro into most mesodermal cell types (cells with characteristics of osteoblasts, chondroblasts, fibroblasts, adipocytes, skeletal, smooth and cardiac myoblasts, endothelial cells), as well as cells with neuroectodermal and with endodermal features. Using retroviral marking we have shown that multi-lineage differentiation is derived from single MAPC. MAPC express active telomerase and can undergo 100+ cell doublings without telomere shortening, suggesting that they do not senesce. MAPC express oct-4 mRNA. MAPC engraft in vivo and persist for 6+ months, differentiate into hematopoietic and epithelial cells in response to local “cues”, and contribute to all somatic cell types when injected in the blastocyst. The finding that stem cells exist in post-natal tissues with previously unknown proliferation and differentiation potential opens up the possibility of using autologous stem cells to treat a host of degenerative, traumatic or congenital diseases.

The only curative therapy for sickle cell disease (SCD) is allogeneic hematopoietic stem cell (HSC) transplantation. Gene therapy approaches for autologous HSC transplantation are being developed. Although earlier engraftment is seen when cells from GCSF-mobilized blood are transplanted than when bone marrow is transplanted, administration of GCSF to patients with SCD can cause significant morbidity. We tested whether primitive hematopoietic progenitors are spontaneously mobilized in the blood of patients with SCD during acute crisis (AC-SCD patients). The frequency of myeloid-lymphoid-initiating cells (ML-ICs) and SCID-repopulating cells (SRCs) was significantly higher in blood from AC-SCD patients than in blood from patients with steady-state SCD or from normal donors. The presence of SRCs in peripheral blood was not associated with detection of long-term culture-initiating cells, consistent with the notion that SRCs are more primitive than long-term culture-initiating cells. As ML-ICs and SRCs were both detected in blood of AC-SCD patients only, these assays may both measure primitive progenitors. The frequency of ML-ICs also correlated with increases in stem cell factor, GCSF, and IL-8 levels in AC-SCD compared with steady-state SCD and normal-donor sera. Because significant numbers of ML-ICs and SRCs are mobilized in the blood without exogenous cytokine treatment during acute crisis of SCD, collection of peripheral blood progenitors during crisis may yield a source of autologous HSCs suitable for ex-vivo correction by gene therapy approaches and subsequent transplantation. ispartof: Journal of Clinical Investigation vol:111 issue:6 pages:811-9 ispartof: location:United States status: published