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Abstract Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53‐independent quiescent state in immature hepatoma cells and in pluripotent stem cell‐derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity‐regulated growth arrest followed by cell maturation, mediated by activation of NF‐κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long‐term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely.

Summary: Interaction with microenvironmental factors is crucial for the regulation of hematopoietic stem cell (HSC) function. Stroma derived factor (SDF)-1α supports HSCs in the quiescent state and is central to the homing of transplanted HSCs. Here, we show that integrin signaling regulates Sdf-1α expression transcriptionally. Systemic deletion of Periostin, an Integrin-αv ligand, showed increased expression of Sdf-1α in bone marrow (BM) niche. Pharmacological inhibition or CRISPR-Cas9-mediated deletion of SRC, resulted in a similar increase in the chemokine expression in vitro. Importantly, systemic SRC-inhibition led to increase in SDF-1α levels in BM plasma. This resulted in a robust increase (14.05 ± 1.22% to 29.11 ± 0.69%) in the homing efficiency of transplanted HSCs. In addition, we observed enhancement in the recovery of blood cell counts following radiation injury, indicating an enhanced hematopoietic function. These results establish a role of SRC-mediated integrin signaling in the transcriptional regulation of Sdf-1α. This mechanism could be harnessed further to improve the hematopoietic function.

Pathological abnormalities in the tau protein give rise to a variety of neurodegenerative diseases, conjointly termed tauopathies. Several tau mutations have been identified in the tau-encoding gene MAPT, affecting either the physical properties of tau or resulting in altered tau splicing. At early disease stages, mitochondrial dysfunction was highlighted with mutant tau compromising almost every aspect of mitochondrial function. Additionally, mitochondria have emerged as fundamental regulators of stem cell function. Here, we show that compared to the isogenic wild-type triple MAPT-mutant human-induced pluripotent stem cells, bearing the pathogenic N279K, P301L, and E10+16 mutations, exhibit deficits in mitochondrial bioenergetics and present altered parameters linked to the metabolic regulation of mitochondria. Moreover, we demonstrate that the triple tau mutations disturb the cellular redox homeostasis and modify the mitochondrial network morphology and distribution. This study provides the first characterization of disease-associated tau-mediated mitochondrial impairments in an advanced human cellular tau pathology model at early disease stages, ranging from mitochondrial bioenergetics to dynamics. Consequently, comprehending better the influence of dysfunctional mitochondria on the development and differentiation of stem cells and their contribution to disease progression may thus assist in the potential prevention and treatment of tau-related neurodegeneration.

Abstract To date, there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell (PSC)-derived) endothelial cells (ECs). Furthermore, the transcriptional programmes driving an LSEC fate have not yet been described. Here, we first present a computational workflow (CenTFinder) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder, we identified several novel LSEC-specific protein markers, such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC TF ETV2) in human PSCs induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers, such as CD32B and MRC1, as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC, as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.

Liver transplantation is currently the only curative therapy for patients with acute or chronic liver failure. However, a dramatic gap between the number of available liver grafts and the number of patients on the transplantation waiting list emphasizes the need for valid liver substitutes. Whole-organ engineering is an emerging field of tissue engineering and regenerative medicine. It aims to generate transplantable and functional organs to support patients on transplantation waiting lists until a graft becomes available. It comprises two base technologies developed in the last decade; (1) organ decellularization to generate a three-dimensional (3D) extracellular matrix scaffold of an organ, and (2) scaffold recellularization to repopulate both the parenchymal and vascular compartments of a decellularized organ. In this review article, recent advancements in both technologies, in relation to liver whole-organ engineering, are presented. We address the potential sources of hepatocytes and non-parenchymal liver cells for repopulation studies, and the role of stem-cell-derived liver progeny is discussed. In addition, different cell seeding strategies, possible graft modifications, and methods used to evaluate the functionality of recellularized liver grafts are outlined. Based on the knowledge gathered from recent transplantation studies, future directions are summarized.

Hepatocytes grown in a dish are immature and do not metabolize compounds as a real liver would. Here, the authors supply stem cell-derived hepatocytes with amino acids at a higher concentration than nutritionally necessary, changing the metabolism of these cells, making them more mature and useful for drug screening and toxicity studies.

Summary: Hematopoietic stem and progenitor cell (HSPC) engraftment after transplantation during anticancer treatment depends on support from the recipient bone marrow (BM) microenvironment. Here, by studying physiological homing of fetal HSPCs, we show the critical requirement of balanced local crosstalk within the skeletal niche for successful HSPC settlement in BM. Transgene-induced overproduction of vascular endothelial growth factor (VEGF) by osteoprogenitor cells elicits stromal and endothelial hyperactivation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSPC homing and survival are drastically impaired. Transcriptome profiling, flow cytometry, and high-resolution imaging indicate alterations in perivascular and endothelial cell characteristics, vascular function and cellular metabolism, associated with increased oxidative stress within the VEGF-enriched BM environment. Thus, developmental HSPC homing to bone is controlled by local stromal-vascular integrity and the oxidative-metabolic status of the recipient milieu. Interestingly, irradiation of adult mice also induces stromal VEGF expression and similar osteo-angiogenic niche changes, underscoring that our findings may contribute targets for improving stem cell therapies.

Frontotemporal dementia (FTD) is a neurodegenerative disease, leading to behavioral changes and language difficulties. Heterozygous loss-of-function mutations in progranulin (GRN) induce haploinsufficiency of the protein and are associated with up to one-third of all genetic FTD cases worldwide. While the loss of GRN is primarily associated with neurodegeneration, the biological functions of the secreted growth factor-like protein are more diverse, ranging from wound healing, inflammation, vasculogenesis, and metabolic regulation to tumor cell growth and metastasis. To date, no disease-modifying treatments exist for FTD, but different therapeutic approaches to boost GRN levels in the central nervous system are currently being developed (including AAV-mediated GRN gene delivery as well as anti-SORT1 antibody therapy). In this review, we provide an overview of the multifaceted regulation of GRN levels and the corresponding therapeutic avenues. We discuss the opportunities, advantages, and potential drawbacks of the diverse approaches. Additionally, we highlight the therapeutic potential of elevating GRN levels beyond patients with loss-of-function mutations in GRN.

The pharmaceutical industry is in high need of efficient and relevant in vitro liver models, which can be incorporated in their drug discovery pipelines to identify potential drugs and their toxicity profiles. Current liver models often rely on cancer cell lines or primary cells, which both have major limitations. However, the development of human induced pluripotent stem cells (hiPSCs) has created a new opportunity for liver disease modeling, drug discovery and liver toxicity research. hiPSCs can be differentiated to any cell of interest, which makes them good candidates for disease modeling and drug discovery. Moreover, hiPSCs, unlike primary cells, can be easily genome-edited, allowing the creation of reporter lines or isogenic controls for patient-derived hiPSCs. Unfortunately, even though liver progeny from hiPSCs has characteristics similar to their in vivo counterparts, the differentiation of iPSCs to fully mature progeny remains highly challenging and is a major obstacle for the full exploitation of these models by pharmaceutical industries. In this review, we discuss current liver-cell differentiation protocols and in vitro iPSC-based liver models that could be used for disease modeling and drug discovery. Furthermore, we will discuss the challenges that still need to be overcome to allow for the successful implementation of these models into pharmaceutical drug discovery platforms.

Summary: Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation. : In this article, García-León JA and colleagues demonstrate the generation of functional oligodendrocytes (OLs) from human pluripotent stem cells in a rapid and efficient manner by the single overexpression of SOX10. Generated OLs resemble primary OLs at the transcriptome level and can myelinate neurons both in vivo and in vitro. Neuron-OL co-cultures, adapted to high-throughput screening formats, responded to drugs affecting myelination. Keywords: oligodendrocyte, induced pluripotent stem cells (iPSCs), multiple sclerosis, amyotrophic lateral sclerosis, myelination, disease modeling, drug screening

Traditional toxicity risk assessment approaches have until recently focussed mainly on histochemical readouts for cell death. Modern toxicology methods attempt to deduce a mechanistic understanding of pathways involved in the development of toxicity, by using transcriptomics and other big data-driven methods such as high-content screening. Here, we used a recently described optimised method to differentiate human induced pluripotent stem cells (hiPSCs) to hepatocyte-like cells (HLCs), to assess their potential to classify hepatotoxic and non-hepatotoxic chemicals and their use in mechanistic toxicity studies. The iPSC-HLCs could accurately classify chemicals causing acute hepatocellular injury, and the transcriptomics data on treated HLCs obtained by TempO-Seq technology linked the cytotoxicity to cellular stress pathways, including oxidative stress and unfolded protein response (UPR). Induction of these stress pathways in response to amiodarone, diclofenac, and ibuprofen, was demonstrated to be concentration and time dependent. The transcriptomics data on diclofenac-treated HLCs were found to be more sensitive in detecting differentially expressed genes in response to treatment, as compared to existing datasets of other diclofenac-treated in vitro hepatocyte models. Hence iPSC-HLCs generated by transcription factor overexpression and in metabolically optimised medium appear suitable for chemical toxicity detection as well as mechanistic toxicity studies.

Abstract 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/FiO2), wet-to-dry weight ratio (W/D) sampling and a multiplex analysis of bronchoalveolar lavage (BAL) (2 × 30 ml) was performed. Results Pulmonary vascular resistance, lung compliance, PaO2/FiO2 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-α, IL-1β and IFN-γ in the BAL, but not in IFN-α; 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.

We derived a stable cell line from Chinese hamster embryonic fibroblasts by transduction of four mouse transcription factors (M3O, Sox2, Klf4, and n-Myc) using a lentiviral vector. The cell line possess all the characteristics of an induced pluripotent stem cell (iPSC) line. Given that Chinese hamster ovary (CHO) cells are the predominant host cells used for therapeutic protein production and no pluripotent stem cell line or other normal cell line has been isolated from Chinese hamster, this iPSC line may serve as a useful tool for research using CHO cells or even be used for deriving new cell lines.

In early mouse pre-implantation development, primitive endoderm (PrE) precursors are platelet-derived growth factor receptor alpha (PDGFRα) positive. Here, we demonstrated that cultured mouse embryonic stem cells (mESCs) express PDGFRα heterogeneously, fluctuating between a PDGFRα+ (PrE-primed) and a platelet endothelial cell adhesion molecule 1 (PECAM1)-positive state (epiblast-primed). The two surface markers can be co-detected on a third subpopulation, expressing epiblast and PrE determinants (double-positive). In vitro, these subpopulations differ in their self-renewal and differentiation capability, transcriptional and epigenetic states. In vivo, double-positive cells contributed to epiblast and PrE, while PrE-primed cells exclusively contributed to PrE derivatives. The transcriptome of PDGFRα+ subpopulations differs from previously described subpopulations and shows similarities with early/mid blastocyst cells. The heterogeneity did not depend on PDGFRα but on leukemia inhibitory factor and fibroblast growth factor signaling and DNA methylation. Thus, PDGFRα+ cells represent the in vitro counterpart of in vivo PrE precursors, and their selection from cultured mESCs yields pure PrE precursors.

Integrins regulate haematopoietic stem cell (HSC) homeostasis and engraftment into the bone marrow (BM) niche upon transplantation. Here, the authors show that HSC quiescence and function in the BM is regulated by the interaction of PERIOSTIN and INTEGRIN αv and subsequent increase in p27Kip1.

Although pluripotent stem cells can be differentiated into the hepatocyte lineages, such cells retain an immature phenotype. As the chromatin state of regulatory regions controls spatiotemporal gene expression during development, we evaluated changes in epigenetic histone marks in lineage-specific genes throughout in vitro hepatocyte differentiation from human embryonic stem cells (hESCs). Active acetylation and methylation marks at promoters and enhancers correlated with progressive changes in gene expression. However, repression-associated H3K27me3 marks at these control regions showed an inverse correlation with gene repression during transition from hepatic endoderm to a hepatocyte-like state. Inhibitor of Enhancer of Zeste Homolog 2 (EZH2) reduced H3K27me3 decoration but did not improve hepatocyte maturation. Thus, H3K27me3 at regulatory regions does not regulate transcription and appears dispensable for hepatocyte lineage differentiation of hESCs in vitro.

Muscular dystrophies are a heterogeneous group of myopathies, characterized by muscle weakness and degeneration, without curative treatment. Mesoangioblasts (MABs) have been proposed as a potential regenerative therapy. To improve our understanding of the in vivo behavior of MABs and the effect of different immunosuppressive therapies, like cyclosporine A or co-stimulation-adhesion blockade therapy, on cell survival noninvasive cell monitoring is required. Therefore, cells were transduced with a lentiviral vector encoding firefly luciferase (Fluc) and the human sodium iodide transporter (hNIS) to allow cell monitoring via bioluminescence imaging (BLI) and small-animal positron emission tomography (PET). Non-H2 matched mMABs were injected in the femoral artery of dystrophic mice and were clearly visible via small-animal PET and BLI. Based on noninvasive imaging data, we were able to show that co-stim was clearly superior to CsA in reducing cell rejection and this was mediated via a reduction in cytotoxic T cells and upregulation of regulatory T cells.

Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

Hematopoietic stem cells (HSCs) in the fetal liver (FL) unlike adult bone marrow (BM) proliferate extensively, posing different metabolic demands. However, metabolic pathways responsible for the production of energy and cellular building blocks in FL HSCs have not been described. Here, we report that FL HSCs use oxygen dependent energy generating pathways significantly more than their BM counterparts. RNA-Seq analysis of E14.5 FL versus BM derived HSCs identified increased expression levels of genes involved in oxidative phosphorylation (OxPhos) and the citric acid cycle (TCA). We demonstrated that FL HSCs contain more mitochondria than BM HSCs, which resulted in increased levels of oxygen consumption and reactive oxygen species (ROS) production. Higher levels of DNA repair and antioxidant pathway gene expression may prevent ROS-mediated (geno)toxicity in FL HSCs. Thus, we here for the first time highlight the underestimated importance of oxygen dependent pathways for generating energy and building blocks in FL HSCs.

Summary: To understand how haploinsufficiency of progranulin (PGRN) causes frontotemporal dementia (FTD), we created induced pluripotent stem cells (iPSCs) from patients carrying the GRNIVS1+5G > C mutation (FTD-iPSCs). FTD-iPSCs were fated to cortical neurons, the cells most affected in FTD. Although generation of neuroprogenitors was unaffected, their further differentiation into CTIP2-, FOXP2-, or TBR1-TUJ1 double-positive cortical neurons, but not motorneurons, was significantly decreased in FTD-neural progeny. Zinc finger nuclease-mediated introduction of GRN cDNA into the AAVS1 locus corrected defects in cortical neurogenesis, demonstrating that PGRN haploinsufficiency causes inefficient cortical neuron generation. RNA sequencing analysis confirmed reversal of the altered gene expression profile following genetic correction. We identified the Wnt signaling pathway as one of the top defective pathways in FTD-iPSC-derived neurons, which was reversed following genetic correction. Differentiation of FTD-iPSCs in the presence of a WNT inhibitor mitigated defective corticogenesis. Therefore, we demonstrate that PGRN haploinsufficiency hampers corticogenesis in vitro. : Verfaillie and colleagues describe the inefficient cortical neuron, but not motorneuron, generation, from FTD-patient-derived iPSCs carrying a mutation in the GRN gene. They show restoration of the defective phenotype following introduction of the GRN cDNA in FTD-iPSC using zinc finger nucleases and by inhibiting the WNT pathway.

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-, atrial-, and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives, which in turn promoted cardiomyocyte differentiation. Moreover, a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation, improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation, measuring action potentials, and intracellular Ca2+ dynamics. These findings are relevant for improving our understanding on human heart development, and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes, similar to those observed in adult cardiac myocytes.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood, aside from numerous congenital abnormalities. FA mouse models have been generated; however, they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero, a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology, we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential, disruption of the second allele causes a severe growth disadvantage. As a result, heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele, quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning, this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage.

Reprogramming can occur by the introduction of key transcription factors (TFs) as well as by epigenetic changes. We demonstrated that histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) combined with a chromatin remodeling medium (CRM) induced expression of a number of definitive endoderm and early and late pancreatic marker genes. When CRM was omitted, endoderm/pancreatic marker genes were not induced. Furthermore, treatment with DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (5AZA) CRM did not affect gene expression changes, and when 5AZA was combined with TSA, no further increase in gene expression of endoderm, pancreatic endoderm, and endocrine markers was seen over levels induced with TSA alone. Interestingly, TSA-CRM did not affect expression of pluripotency and hepatocyte genes but induced some mesoderm transcripts. Upon removal of TSA-CRM, the endoderm/pancreatic gene expression profile returned to baseline. Our findings underscore the role epigenetic modification in transdifferentiation of one somatic cell into another. However, full reprogramming of fibroblasts to β-cells will require combination of this approach with TF overexpression and/or culture of the partially reprogrammed cells under β-cell specific conditions.

Numerous neurodegenerative and neuromuscular disorders are associated with cell-specific depletion in the human body. This imbalance in tissue homeostasis is in healthy individuals repaired by the presence of endogenous stem cells that can replace the lost cell type. However, in most disorders, a genetic origin or limited presence or exhaustion of stem cells impairs correct cell replacement. During the last 30 years, methods to readily isolate and expand stem cells have been developed and this resulted in a major change in the regenerative medicine field as it generates sufficient amount of cells for human transplantation applications. Furthermore, stem cells have been shown to release cytokines with beneficial effects for several diseases. At present however, clinical stem cell transplantations studies are struggling to demonstrate clinical efficacy despite promising preclinical results. Therefore, to allow stem cell therapy to achieve its full potential, more insight in their in vivo behavior has to be achieved. Different methods to noninvasively monitor these cells have been developed and are discussed. In some cases, stem cell monitoring even reached the clinical setting. We anticipate that by further exploring these imaging possibilities and unraveling their in vivo behavior further improvement in stem cell transplantations will be achieved.

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.

Human multipotent adult progenitor cells (hMAPCs) are isolated from bone marrow with a more extensive expansion capacity compared to human mesenchymal stem cells (hMSCs) and with the ability to differentiate into endothelium. Like hMSCs, hMAPCs inhibit T-cell proliferation induced by alloantigens. In this study, we tested the interaction between hMAPCs and natural killer (NK) cells. We assessed the susceptibility of hMAPCs to NK cell-mediated lysis and the immunomodulation of hMAPCs on NK cell function during IL-2-driven stimulation and the cytolytic effector phase. Human MAPCs express the ligands PVR and ULBP-2/5/6, which are recognized by activating NK cell receptors. However, they also express MHC class I molecules, which induce inhibitory signals in NK cells. Freshly isolated NK cells at different effector:target ratios did not kill hMAPCs as assessed by an MTT and 51 Cr-release assay, while hMAPCs impaired the cytotoxic activity of resting NK cells against the NK-sensitive K562 leukemia cell line. By contrast, IL-2-stimulated NK cells were capable of killing hMAPCs, and preactivated NK cells were not influenced during their cytotoxic effector function against K562 cells by hMAPCs. When added during the 6-day preactivation phase with IL-2, hMAPCs dose-dependently reduced NK cell proliferation in an IDO-dependent manner, but they did not influence the induction of cytotoxic capacity by IL-2. This study indicates that human MAPCs mutually interact with NK cells.

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohn's disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.

In this review, we discuss the migration and homing ability of mesenchymal stem cells (MSCs) and MSC-like cells and factors influencing this. We also discuss studies related to the mechanism of migration and homing and the approaches undertaken to enhance it. Finally, we describe the different methods available and frequently used to track and identify the injected cells in vivo.

Pleomorphic adenoma gene-like 1 (PLAGL1) has been linked to transient neonatal diabetes mellitus. Here, we investigated the role of the related pleomorphic adenoma gene 1 (PLAG1) in glucose homeostasis. PLAG1 transgenic mice in which expression of the PLAG1 transgene can be targeted to different organs by Cre-mediated modulation were crossed with Pdx1-Cre or Ngn3-Cre mice, resulting in double transgenic P1-Pdx1Cre or P1-Ngn3Cre mice, respectively. P1-Pdx1Cre and P1-Ngn3Cre mice developed hyperplasia of pancreatic islets due to increased β- and δ- but not α-cell proliferation. In young P1-Pdx1Cre mice (less than 15 weeks) there was a balanced increase in the pancreatic content of insulin and somatostatin, which was associated with normoglycemia. In older P1-Pdx1Cre mice the pancreatic somatostatin content far exceeded that of insulin, leading to the progressive development of severe hypoglycemia beyond 30 weeks. In contrast, in older P1-Ngn3Cre mice the relative increase of the pancreatic insulin content exceeded that of somatostatin and these mice remained normoglycemic. In conclusion, forced expression of PLAG1 under the control of the Pdx1 or Ngn3 promoter in murine pancreas induces different degrees of endocrine hormone imbalances within the pancreas, which is associated with hypoglycemia in P1-Pdx1Cre mice but not P1-Ngn3Cre mice. These results suggest that once stem cell-derived islet transplantations become possible, the appropriate balance between different hormone-producing cells will need to be preserved to prevent deregulated glucose metabolism.

Non-viral integrating systems, PhiC31 phage integrase (ϕC31), and Sleeping Beauty transposase (SB), provide an effective method for ex vivo gene delivery into cells. Here, we used a plasmid-encoding GFP and neomycin phosphotransferase along with recognition sequences for both ϕC31 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 to ϕC31. Sequence identification of chromosomal junction sites indicated a random profile for SB-mediated integrants and a more restricted profile for ϕC31 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 and ϕC31 are effective non-viral integrating systems for genetic engineering of MAPC in basic studies of stem cell biology.

The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival, and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell-based therapies. To follow the fate of grafted cells in vivo, a number of noninvasive imaging approaches have been developed. Magnetic resonance imaging (MRI) is a high-resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of three different stem cell populations [mouse embryonic stem cells (mESC), rat multipotent adult progenitor cells (rMAPC), and mouse mesenchymal stem cells (mMSC)] with three different (ultra)small superparamagnetic iron oxide [(U)SPIO] particles (Resovist®, Endorem®, Sinerem®) was compared. Labeling efficiency with Resovist® and Endorem® differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI-based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.

Previously we reported the characterization of multipotent adult progenitor cells (MAPCs) isolated from the bone marrow of rodents. In that study, single murine MAPCs derived from ROSA-26, β-galactosidase (β-Gal)-positive transgenic mice were injected into E3.5 C57/Bl6 mouse blastocysts. The resultant chimeric blastocysts were then implanted into pseudopregnant females and were allowed to develop naturally through birth and into adulthood. Chimeric mice were sacrificed 6 to 20 weeks after birth, and were processed for histological analysis. β-Galactosidase activity was identified in all organs and tissues examined, and tissuespecific differentiation and engraftment was confirmed by colabeling with antibodies that recognize β-Gal and tissue-specific markers. In the present study we have examined neural engraftment derived from the clonal expansion of a single MAPC during rodent development, and characterized the neural phenotype of MAPCs in the resultant chimeric animals. Donor cell-derived β-Gal activity was evident throughout the brain. Double and triple immunofluorescent labeling studies revealed MAPC-derived neurons (NeuN/β-Gal) and astrocytes (GFAP/β-Gal) in the cortex, striatum, medial septal nucleus, hippocampus, cerebellum, substantia nigra, and thalamus. More specifically, donor-derived neurons contributed to each of the cellular layers of the cortex; the pyramidal and granule cell layers, as well as the hilus, of the hippocampus; Purkinje and granule cell layers in the cerebellum; and GABAergic cells in the caudate and putamen. This study haracterizes the potential for MAPCs to differentiate into specific neuronal and glial phenotypes, and to integrate normally during development, after implantation into blastocysts, and provides additional evidence that MAPCs exhibit properties similar to embryonic stem cells.