Vδ1 T cells integrated in full thickness skin equivalents: a model for the role of human skin-resident γδT cells.

Vδ1 T cells are a subpopulation of γδT cells found in human dermis. In contrast to murine skin-resident γδT cells, much less is known regarding their role and function in skin health and disease. Here we report the successful integration of Vδ1 T cells into long-term fibroblast-derived matrix skin equivalents (SE). We isolated Vδ1 T cells from human blood, where they are rare, and established conditions for the integration and maintenance of the freshly isolated Vδ1 T cells in the SEs. Plated on top of the dermal equivalents (DEs), almost all Vδ1 T cells migrated into the dermal matrix where they exerted their influence on both the DE and the epithelium. Vδ1 T cells contributed to epidermal differentiation as indicated by histology, expression of epidermal differentiation markers and RNAseq expression profile. When complemented with the carcinoma-derived SCC13 cells instead of HaCaT, our data suggest a role for Vδ1 T cells in slowing growth of the tumor cells, as indicated by reduced stratification and changes in gene expression profiles. Together, we demonstrate the successful establishment of human Vδ1 T cell-competent skin and skin carcinoma equivalents (SE, SCE) and provide evidence for molecular and functional consequences of the Vδ1 T cells on their respective environment.

Hepatic stellate cells: current state and open questions.

This review article summarizes 20 years of our research on hepatic stellate cells within the framework of two collaborative research centers CRC575 and CRC974 at the Heinrich Heine University. Over this period, stellate cells were identified for the first time as mesenchymal stem cells of the liver, and important functions of these cells in the context of liver regeneration were discovered. Furthermore, it was determined that the space of Disse - bounded by the sinusoidal endothelium and hepatocytes - functions as a stem cell niche for stellate cells. Essential elements of this niche that control the maintenance of hepatic stellate cells have been identified alongside their impairment with age. This article aims to highlight previous studies on stellate cells and critically examine and identify open questions and future research directions.

Improved Recovery from Liver Fibrosis by Crenolanib.

Chronic liver diseases are associated with excessive deposition of extracellular matrix proteins. This so-called fibrosis can progress to cirrhosis and impair vital functions of the liver. We examined whether the receptor tyrosine kinase (RTK) class III inhibitor Crenolanib affects the behavior of hepatic stellate cells (HSC) involved in fibrogenesis. Rats were treated with thioacetamide (TAA) for 18 weeks to trigger fibrosis. After TAA treatment, the animals received Crenolanib for two weeks, which significantly improved recovery from liver fibrosis. Because Crenolanib predominantly inhibits the RTK platelet-derived growth factor receptor-ß, impaired HSC proliferation might be responsible for this beneficial effect. Interestingly, blocking of RTK signaling by Crenolanib not only hindered HSC proliferation but also triggered their specification into hepatic endoderm. Endodermal specification was mediated by p38 mitogen-activated kinase (p38 MAPK) and c-Jun-activated kinase (JNK) signaling; however, this process remained incomplete, and the HSC accumulated lipids. JNK activation was induced by stress response-associated inositol-requiring enzyme-1α (IRE1α) in response to Crenolanib treatment, whereas ß-catenin-dependent WNT signaling was able to counteract this process. In conclusion, the Crenolanib-mediated inhibition of RTK impeded HSC proliferation and triggered stress responses, initiating developmental processes in HSC that might have contributed to improved recovery from liver fibrosis in TAA-treated rats.

Impaired integrin α5 /ß1 -mediated hepatocyte growth factor release by stellate cells of the aged liver.

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α5 /ß1 is an important mechanosensor in HSC involved in shear stress-induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α5 and ß1 decreases in liver and HSC from aged rats. CRISPR/Cas9-mediated integrin α5 and ß1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α5 and laminin gene expression whereas integrin ß1 remains unaffected. In the aged liver, laminin ß2 and γ1 protein chains as components of laminin-521 are lowered. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin-521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence-associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α5 /ß1 in HSC contributes to age-related reduction of ECM and HGF release that could affect liver regeneration.

Prominin-1 (CD133) modulates the architecture and dynamics of microvilli.

Prominin-1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non-epithelial cells. Irrespective of the cell type, prominin-1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin-Darby canine kidney cells as the main model. Our high-resolution analysis revealed that upon the overexpression of prominin-1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob-like morphologies were observed and stimulated by mutations in the ganglioside-binding site of prominin-1. The altered phenotypes were caused by the interaction of prominin-1 with phosphoinositide 3-kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin-1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin-1-ganglioside membrane complexes, phosphoinositide 3-kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin-1 and its mutants modified the structure of filopodia emerging from fibroblast-like cells and silencing human prominin-1 in primary hematopoietic stem cells resulted in the loss of uropod-associated microvilli. Altogether, these findings strengthen the role of prominin-1 as an organizer of cellular protrusions.

Transplanted Human Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Support Liver Regeneration in Gunn Rats.

Gunn rats bear a mutation within the uridine diphosphate glucuronosyltransferase-1a1 (Ugt1a1) gene resulting in high serum bilirubin levels as seen in Crigler-Najjar syndrome. In this study, the Gunn rat was used as an animal model for heritable liver dysfunction. Induced mesenchymal stem cells (iMSCs) derived from embryonic stem cells (H1) and induced pluripotent stem cells were transplanted into Gunn rats after partial hepatectomy. The iMSCs engrafted and survived in the liver for up to 2 months. The transplanted iMSCs differentiated into functional hepatocytes as evidenced by partially suppressed hyperbilirubinemia and expression of multiple human-specific hepatocyte markers such as albumin, hepatocyte nuclear factor 4α, UGT1A1, cytokeratin 18, bile salt export pump, multidrug resistance protein 2, Na/taurocholate-cotransporting polypeptide, and α-fetoprotein. These findings imply that transplanted human iMSCs can contribute to liver regeneration in vivo and thus represent a promising tool for the treatment of inherited liver diseases.

Tunneling nanotubes mediate the transfer of stem cell marker CD133 between hematopoietic progenitor cells.

Deciphering all mechanisms of intercellular communication used by hematopoietic progenitors is important, not only for basic stem cell research, but also in view of their therapeutic relevance. Here, we investigated whether these cells can produce the thin F-actin-based plasma membrane protrusions referred to as tunneling nanotubes (TNTs), which are known to bridge cells over long distances without contact with the substratum and transfer cargo molecules along them in various biological processes. We found that human primary CD34+ hematopoietic progenitors and leukemic KG1a cells develop such structures upon culture on primary mesenchymal stromal cells or specific extracellular-matrix-based substrata. Time-lapse video microscopy revealed that cell dislodgement is the primary mechanism responsible for TNT biogenesis. Surprisingly, we found that, among various cluster of differentiation (CD) markers, only the stem cell antigen CD133 is transferred between cells. It is selectively and directionally transported along the surface of TNTs in small clusters, such as cytoplasmic phospho-myosin light chain 2, suggesting that the latter actin motor protein might be implicated in this process. Our data provide new insights into the biology of hematopoietic progenitors that can contribute to our understanding of all facets of intercellular communication in the bone marrow microenvironment under healthy or cancerous conditions.

Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells.

Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34(+) hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34(+)CD133(-) HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34(+)CD133(+) and CD133(+)CD34(-) phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.

CD133 is a modifier of hematopoietic progenitor frequencies but is dispensable for the maintenance of mouse hematopoietic stem cells.

Pentatransmembrane glycoprotein prominin-1 (CD133) is expressed at the cell surface of multiple somatic stem cells, and it is widely used as a cell surface marker for the isolation and characterization of human hematopoietic stem cells (HSCs) and cancer stem cells. CD133 has been linked on a cell biological basis to stem cell-fate decisions in human HSCs and emerges as an important physiological regulator of stem cell maintenance and expansion. Its expression and physiological relevance in the murine hematopoietic system is nevertheless elusive. We show here that CD133 is expressed by bone marrow-resident murine HSCs and myeloid precursor cells with the developmental propensity to give rise to granulocytes and monocytes. However, CD133 is dispensable for the pool size and function of HSCs during steady-state hematopoiesis and after transplantation, demonstrating a substantial species difference between mouse and man. Blood cell numbers in the periphery are normal; however, CD133 appears to be a modifier for the development of growth-factor responsive myeloerythroid precursor cells in the bone marrow under steady state and mature red blood cells after hematopoietic stress. Taken together, these studies show that CD133 is not a critical regulator of hematopoietic stem cell function in mouse but that it modifies frequencies of growth-factor responsive hematopoietic progenitor cells during steady state and after myelotoxic stress in vivo.