Single-Cell RNA Sequencing Reveals Repair Features of Human Umbilical Cord Mesenchymal Stromal Cells.

RATIONALE: The chronic lung disease bronchopulmonary dysplasia (BPD) is the most severe complication of extreme prematurity. BPD results in impaired lung alveolar and vascular development and long-term respiratory morbidity, for which only supportive therapies exist. Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) improve lung structure and function in experimental BPD. Results of clinical trials with MSCs for many disorders do not yet match the promising preclinical studies. A lack of specific criteria to define functionally distinct MSCs persists. OBJECTIVES: To determine and correlate single-cell UC-MSC transcriptomic profile with therapeutic potential. METHODS: UC-MSCs from five term donors and human neonatal dermal fibroblasts (HNDFs, control cells of mesenchymal origin) transcriptomes were investigated by single-cell RNA sequencing analysis (scRNA-seq). The lung-protective effect of UC-MSCs with a distinct transcriptome and control HNDFs was tested in vivo in hyperoxia-induced neonatal lung injury in rats. MEASUREMENTS AND MAIN RESULTS: UC-MSCs showed limited transcriptomic heterogeneity, but were different from HNDFs. Gene ontology enrichment analysis revealed distinct - progenitor-like and fibroblast-like - UC-MSC subpopulations. Only the treatment with progenitor-like UC-MSCs improved lung function and structure and attenuated pulmonary hypertension in hyperoxia-exposed rat pups. Moreover, scRNA-seq identified major histocompatibility complex class I as a molecular marker of non-therapeutic cells and associated with decreased lung retention. CONCLUSIONS: UC-MSCs with a progenitor-like transcriptome, but not with a fibroblast-like transcriptome, provide lung protection in experimental BPD. High expression of major histocompatibility complex class I is associated with reduced therapeutic benefit. scRNA-seq may be useful to identify subsets of MSCs with superior repair capacity for clinical application.

Prophylactic Administration of Mesenchymal Stromal Cells Does Not Prevent Arrested Lung Development in Extremely Premature-Born Non-Human Primates.

Premature birth is a leading cause of childhood morbidity and mortality and often followed by an arrest of postnatal lung development called bronchopulmonary dysplasia. Therapies using exogenous mesenchymal stromal cells (MSC) have proven highly efficacious in term-born rodent models of this disease, but effects of MSC in actual premature-born lungs are largely unknown. Here, we investigated thirteen non-human primates (baboons; Papio spp.) that were born at the limit of viability and given a single, intravenous dose of ten million human umbilical cord tissue-derived MSC per kilogram or placebo immediately after birth. Following two weeks of human-equivalent neonatal intensive care including mechanical ventilation, lung function testing and echocardiographic studies, lung tissues were analyzed using unbiased stereology. We noted that therapy with MSC was feasible, safe and without signs of engraftment when administered as controlled infusion over 15 minutes, but linked to adverse events when given faster. Administration of cells was associated with improved cardiovascular stability, but neither benefited lung structure, nor lung function after two weeks of extrauterine life. We concluded that a single, intravenous administration of MSC had no short- to mid-term lung-protective effects in extremely premature-born baboons, sharply contrasting data from term-born rodent models of arrested postnatal lung development and urging for investigations on the mechanisms of cell-based therapies for diseases of prematurity in actual premature organisms.

Honest Signals of Status: Facial and Bodily Dominance Are Related to Success in Physical but Not Nonphysical Competition.

Recent studies suggest that both facial and bodily dominance promote high status positions and predict status-seeking behaviors such as aggression and social dominance. An evolutionarily relevant context in which associations between these dominance signals and status outcomes may be prevalent are face-to-face status contests. The present study examined whether facial and bodily dominance predicted success in dyadic competitions (one physical discipline, arm wrestling, and three nonphysical disciplines) in men (N = 125) in a controlled laboratory setting. Men's bodies and faces were independently rated for physical dominance, and associations of these ratings with contest outcomes as well as mediating and moderating variables (such as physical strength, body height, trait dominance, baseline and reactive testosterone) were examined. Both facial and bodily dominance positively predicted success in the physical discipline, mediated by physical strength, but not in the three nonphysical disciplines. Our findings demonstrate that facial and bodily physical dominance may be honest signals for men's formidability and hence status potential, at least in a physically competitive context.

Automated Clinical Grade Expansion of Regulatory T Cells in a Fully Closed System.

Adoptive transfer of T regulatory cells (Treg) has been successfully exploited in the context of graft-versus-host disease, transplantation, and autoimmune disease. For the majority of applications, clinical administration of Treg requires laborious ex vivo expansion and typically involves open handling for culture feeds and repetitive sampling. Here we show results from our approach to translate manual Treg manufacturing to the fully closed automated CliniMACS Prodigy® system reducing contamination risk, hands-on time, and quality variation from human intervention. Polyclonal Treg were isolated from total nucleated cells obtained through leukapheresis of healthy donors by CD8+ cell depletion and subsequent CD25high enrichment. Treg were expanded with the CliniMACS Prodigy® device using clinical-grade cell culture medium, rapamycin, IL-2, and αCD3/αCD28 beads for 13-14 days. We successfully integrated expansion bead removal and final formulation into the automated procedure, finalizing the process with a ready to use product for bedside transfusion. Automated Treg expansion was conducted in parallel to an established manual manufacturing process using G-Rex cell culture flasks. We could prove similar expansion kinetics leading to a cell yield of up to 2.12 × 109 cells with the CliniMACS Prodigy® and comparable product phenotype of >90% CD4+CD25highCD127lowFOXP3+ cells that had similar in vitro immunosuppressive function. Efficiency of expansion bead depletion was comparable to the CliniMACS® Plus system and the final ready-to-infuse product had phenotype stability and high vitality after overnight storage. We anticipate this newly developed closed system expansion approach to be a starting point for the development of enhanced throughput clinical scale Treg manufacture, and for safe automated generation of antigen-specific Treg grafted with a chimeric antigen receptor (CAR Treg).

Oxygen Disrupts Human Fetal Lung Mesenchymal Cells. Implications for Bronchopulmonary Dysplasia.

Exogenous mesenchymal stromal cells (MSCs) ameliorate experimental bronchopulmonary dysplasia. Moreover, data from term-born animal models and human tracheal aspirate-derived cells suggest altered mesenchymal signaling in the pathophysiology of neonatal lung disease. We hypothesized that hyperoxia, a factor contributing to the development of bronchopulmonary dysplasia, perturbs human lung-resident MSC function. Mesenchymal cells were isolated from human fetal lung tissue (16-18 wk of gestation), characterized and cultured in conditions resembling either intrauterine (5% O2) or extrauterine (21% and 60% O2) atmospheres. Secretome data were compared with MSCs obtained from term umbilical cord tissues. The human fetal lung mesenchyme almost exclusively contains CD146pos. MSCs expressing SOX-2 and OCT-4, which secrete elastin, fibroblast growth factors 7 and 10, vascular endothelial growth factor, angiogenin, and other lung cell-protecting/-maturing proteins. Exposure to extrauterine atmospheres in vitro leads to excessive proliferation, reduced colony-forming ability, alterations in the cell's surface marker profile, decreased elastin deposition, and impaired secretion of factors important for lung growth. Conversely, umbilical cord-derived MSCs abundantly secreted factors that impaired lung MSCs are unable to produce. Oxygen-impaired human fetal lung MSC function may contribute to disrupted repair capacity and arrested lung growth. Exogenous MSCs may act by triggering the signaling pathways lost by impaired endogenous lung mesenchymal cells.

Microarray analysis for delineating the gene expression in biopsies of gastrocnemius muscle of patients with chronic critical limb ischaemia compared with non-ischaemic controls.

BACKGROUND: Microarray analysis has been carried out in this pilot study to compare delineated gene expression profiles in the biopsies of skeletal muscle taken from patients with chronic critical limb ischaemia (CLI) and non-ischaemic control subjects. PATIENTS AND METHODS: Biopsy of gastrocnemius muscle was obtained from six patients with unreconstructed CLI referred for surgical major amputation. As control, biopsies of six patients undergoing elective knee arthroplasty without evidence of peripheral arterial occlusive disease were taken. The differences in gene expression associated with angiogenic processes in specimens obtained from ischaemic and non-ischaemic skeletal muscle were confirmed by quantitative real-time polymerase chain reaction (PCR) analysis. RESULTS: Compared with non-ischaemic skeletal muscle biopsy of chronic-ischaemic skeletal muscle contained 55 significantly up-regulated and 45 down-regulated genes, out of which 64 genes had a known genetic product. Tissue samples of ischaemic muscle were characterized by increased expression of cell survival factors (e. g. tissue factor pathway inhibitor 2) in combination with reduced expression of cell proliferation effectors (e. g. microfibrillar-associated protein 5 and transferrin receptor). The expression of growth factors (e. g. early growth response 3 and chemokine receptor chemokine C-X-C motif ligand 4) which play a central role in arterial and angiogenic processes and anti-angiogenetic factors (e. g. pentraxin 3) were increased in chronic ischaemic skeletal muscle. An increased expression of extracellular matrix proteins (e. g. cysteine-rich angiogenic inducer 61) was also observed. CONCLUSIONS: Gene expression profiles in biopsies of gastrocnemius muscle in patients with chronic critical limb ischaemia showed an increase in pro-survival factors, extracellular matrix protein deposition, and impaired proliferation, compared with non-ischaemic controls. Further studies are required to analyse the endogenous repair mechanism.

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.

Haematopoietic stem cell differentiation promotes the release of prominin-1/CD133-containing membrane vesicles--a role of the endocytic-exocytic pathway.

The differentiation of stem cells is a fundamental process in cell biology and understanding its mechanism might open a new avenue for therapeutic strategies. Using an ex vivo co-culture system consisting of human primary haematopoietic stem and progenitor cells growing on multipotent mesenchymal stromal cells as a feeder cell layer, we describe here the exosome-mediated release of small membrane vesicles containing the stem and cancer stem cell marker prominin-1 (CD133) during haematopoietic cell differentiation. Surprisingly, this contrasts with the budding mechanism underlying the release of this cholesterol-binding protein from plasma membrane protrusions of neural progenitors. Nevertheless, in both progenitor cell types, protein-lipid assemblies might be the essential structural determinant in the release process of prominin-1. Collectively, these data support the concept that prominin-1-containing lipid rafts may host key determinants necessary to maintain stem cell properties and their quantitative reduction or loss may result in cellular differentiation.

Polarization and migration of hematopoietic stem and progenitor cells rely on the RhoA/ROCK I pathway and an active reorganization of the microtubule network.

Understanding the physiological migration of hematopoietic progenitors is important, not only for basic stem cell research, but also in view of their therapeutic relevance. Here, we investigated the role of the Rho kinase pathway in the morphology and migration of hematopoietic progenitors using an ex vivo co-culture consisting of human primary CD34(+) progenitors and mesenchymal stromal cells. The addition of the Rho kinase inhibitor Y-27632 led to the abolishment of the uropod and microvillar-like structures of hematopoietic progenitors, concomitant with a redistribution of proteins found therein (prominin-1 and ezrin). Y-27632-treated cells displayed a deficiency in migration. Time-lapse video microscopy revealed impairment of the rear pole retraction. Interestingly, the knockdown of ROCK I, but not ROCK II, using RNA interference (RNAi) was sufficient to cause the referred morphological and migrational changes. Unexpectedly, the addition of nocodazole to either Y-27632- or ROCK I RNAi-treated cells could restore their polarized morphology and migration suggesting an active role for the microtubule network in tail retraction. Finally, we could demonstrate using RNAi that RhoA, the upstream regulator of ROCK, is involved in these processes. Collectively, our data provide new insights regarding the role of RhoA/ROCK I and the microtubules in the migration of stem cells.

Differential expression of biofunctional GM1 and GM3 gangliosides within the plastic-adherent multipotent mesenchymal stromal cell population.

BACKGROUND AIMS: It is unclear whether the plastic-adherent multipotent mesenchymal stromal cells (MSC) isolated from human bone marrow (BM) represent a uniform cell population or are heterogeneous in terms of cell-surface constituents and hence functionality. METHODS: We investigated the expression profile of certain biofunctional lipids by plastic-adherent MSC, focusing particularly on two membrane microdomain (lipid raft)-associated monosialogangliosides, GM1 and GM3, using indirect confocal laser scanning fluorescence microscopy and flow cytometry. RESULTS: Phenotypically, we observed a differential expression where certain MSC subsets exhibited GM1, GM3 or both at the plasma membrane. Furthermore, disialoganglioside GD2 detection increased the complexity of the expression patterns, giving rise to seven identifiable cell phenotypes. Variation of standard culture conditions, such as the number of cell passage and period in culture, as well as donors, did not influence the heterologous ganglioside expression profile. In contrast, the binding of various lectins appeared homogeneous throughout the MSC population, indicating that the general glycosylation pattern remained common. Morphologically, the expression of a given ganglioside-based phenotype was not related to a cell with particular size or shape. Interestingly, a segregation of GM1 and GM3 clusters was observed, GM3 being mostly excluded from the highly curved plasma membrane protrusions. CONCLUSIONS: These data highlight the phenotypic heterogeneity of plastic-adherent MSC in terms of certain lipid constituents of the plasma membrane, and the presence and/or absence of distinct ganglioside-based membrane microdomains suggest their potential functional diversity.

The stem cell marker CD133 (Prominin-1) is expressed in various human glandular epithelia.

Human prominin-1 (CD133) is expressed by various stem and progenitor cells originating from diverse sources. In addition to stem cells, its mouse ortholog is expressed in a broad range of adult epithelial cells, where it is selectively concentrated in their apical domain. The lack of detection of prominin-1 in adult human epithelia might be explained, at least in part, by the specificity of the widely used AC133 antibody, which recognizes an epitope that seems dependent on glycosylation. Here we decided to re-examine its expression in adult human tissues, particularly in glandular epithelia, using a novel monoclonal antibody (80B258) generated against the human prominin-1 polypeptide. In examined tissues, we observed 80B258 immunoreactivity at the apical or apicolateral membranes of polarized cells. For instance, we found expression in secretory serous and mucous cells as well as intercalated ducts of the large salivary and lacrimal glands. In sweat glands including the gland of Moll, 80B258 immunoreactivity was found in the secretory (eccrine and apocrine glands) and duct (eccrine glands) portion. In the liver, 80B258 immunoreactivity was identified in the canals of Hering, bile ductules, and small interlobular bile ducts. In the uterus, we detected 80B258 immunoreactivity in endometrial and cervical glands. Together these data show that the overall expression of human prominin-1 is beyond the rare primitive cells, and it seems to be a general marker of apical or apicolateral membrane of glandular epithelia. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

New insights into the cell biology of hematopoietic progenitors by studying prominin-1 (CD133).

Prominin-1 (alias CD133) has received considerable interest because of its expression by several stem and progenitor cells originating from various sources, including the neural and hematopoietic systems. As a cell surface marker, prominin-1 is now used for somatic stem cell isolation. Its expression in cancer stem cells has broadened its clinical value, as it might be useful to outline new prospects for more effective cancer therapies by targeting tumor-initiating cells. Cell biological studies of this molecule have demonstrated that it is specifically concentrated in various membrane structures that protrude from the planar areas of the plasmalemma. Prominin-1 binds to the plasma membrane cholesterol and is associated with a particular membrane microdomain in a cholesterol-dependent manner. Although its physiological function is not yet determined, it is becoming clear that this cell surface protein, as a unique marker of both plasma membrane protrusions and membrane microdomains, might reveal new aspects of the cell biology of rare stem and cancer stem cells. The aim of this review is to outline the recent discoveries regarding the dynamic reorganization of the plasma membrane of rare CD133+ hematopoietic progenitor cells during cell migration and division.

Polarization of human hematopoietic progenitors during contact with multipotent mesenchymal stromal cells: effects on proliferation and clonogenicity.

Establishment of a defined cell culture system that facilitates ex vivo expansion of isolated hematopoietic stem and progenitor cells (HSPCs) is a crucial issue in hematology and stem cell transplantation. Here we have evaluated the capacity of primary human multipotent mesenchymal stromal cells (MSCs) to support the ex vivo expansion of peripheral CD34(+)-enriched HSPCs. We observed that HSPCs co-cultured on MSCs showed a substantially higher total expansion rate compared to those growing without. Moreover, in addition to the expansion of CD34(+)CD133(+) and CD34(+)CD133(-) cells, a third population of CD133(+)CD34(-) stem cells became detectable after expansion. Direct contact between HSPCs and the feeder layer appears beneficial for the expansion of HSPCs harboring CD133(+) phenotype, i.e., CD34(+)CD133(+) and CD133(+)CD34(-), in contrast to CD34(+)CD133(-) cells. Interestingly, electron microscopy and immunofluorescence analyses revealed that adherent HSPCs display various morphologies; they are either round with, in some cases, the appearance of a microvillar pole or exhibit several distinct types of plasma membrane protrusions such as lamellipodium and magnupodium. CD133 is selectively concentrated therein, whereas CD34 is randomly distributed over the entire surface of HSPCs. Together, this co-culture offers a unique experimental system to further characterize the biology and role of markers of rare stem cell populations.

Prominin-1/CD133, a neural and hematopoietic stem cell marker, is expressed in adult human differentiated cells and certain types of kidney cancer.

Human prominin-1/CD133 has been reported to be expressed in neural and hematopoietic stem/progenitor cells and in embryonic, but not adult, epithelia. This lack of detection of human prominin-1, as defined by its glycosylation-dependent AC133 epitope, is surprising given the expression of the murine ortholog in adult epithelia. Here, we demonstrate, by using a novel prominin-1 antiserum (alphahE2), that the decrease of AC133 immunoreactivity observed during differentiation of the colonic adenocarcinoma-derived Caco-2 cells is not paralleled by a down-regulation of prominin-1. We have also shown that alphahE2 immunoreactivity, but not AC133 immunoreactivity, is present in several adult human tissues, such as kidney proximal tubules and the parietal layer of Bowman's capsule of juxtamedullary nephrons, and in lactiferous ducts of the mammary gland. These observations suggest that only the AC133 epitope is down-regulated upon cell differentiation. Furthermore, alphahE2 immunoreactivity has been detected in several kidney carcinomas derived from proximal tubules, independent of their grading. Interestingly, in one particular case, the AC133 epitope, which is restricted to stem cells in normal adult tissue, was up-regulated in the vicinity of the tumor. Our data thus show that (1) in adults, the expression of human prominin-1 is not limited to stem and progenitor cells, and (2) the epitopes of prominin-1 might be useful for investigating solid cancers.

Segregation of lipid raft markers including CD133 in polarized human hematopoietic stem and progenitor cells.

During ontogenesis and the entire adult life hematopoietic stem and progenitor cells have the capability to migrate. In comparison to the process of peripheral leukocyte migration in inflammatory responses, the molecular and cellular mechanisms governing the migration of these cells remain poorly understood. A common feature of migrating cells is that they need to become polarized before they migrate. Here we have investigated the issue of cell polarity of hematopoietic stem/progenitor cells in detail. We found that human CD34(+) hematopoietic cells (1) acquire a polarized cell shape upon cultivation, with the formation of a leading edge at the front pole and a uropod at the rear pole; (2) exhibit an amoeboid movement, which is similar to the one described for migrating peripheral leukocytes; and (3) redistribute several lipid raft markers including cholesterol-binding protein prominin-1 (CD133) in specialized plasma membrane domains. Furthermore, polarization of CD34(+) cells is stimulated by early acting cytokines and requires the activity of phosphoinositol-3-kinase as previously reported for peripheral leukocyte polarization. Together, our data reveal a strong correlation between polarization and migration of peripheral leukocytes and hematopoietic stem/progenitor cells and suggest that they are governed by similar mechanisms.

MDR1 gene polymorphisms affect therapy outcome in acute myeloid leukemia patients.

Allelic variants of the MDR-1 gene have been shown recently to influence protein expression and P-glycoprotein (P-gp) function in healthy volunteers. Therefore, 405 acute myeloid leukemia patients were investigated for somatic genotypes of the three most frequent single nucleotide polymorphisms (SNPs) in exons 12, 21, and 26. In all three loci, homozygous wild-type alleles were classified as genotype A, heterozygous as B, and homozygous mutant (alternative) allele as C. Patients with the C genotype in exons 12 and 26 showed a lower median age (both P < 0.05). Additionally, the C genotype in exons 12 and 26 was associated with cytogenetic poor risk aberrations (both P < 0.05). A possible regulatory impact of the SNPs on MDR1 mRNA expression was investigated by a Real time-PCR assay. MDR1 expression was strongly correlated with a decreased complete remission rate (P = 0.01) but failed to predict decreased overall survival (OS). There was a significant association of the A genotype in exons 21 (P = 0.05) and 26 (P < 0.05) with lower MDR1 expression, whereas the B variants showed highest MDR1 values at all three investigated gene loci. The A genotype in exon 26 was associated with lower OS (P < 0,01). In these patients, worse OS is likely attributable to an increased risk of relapse (P < 0.001). We were able to detect a linkage disequilibrium of the investigated SNPs, indicating combined polymorphisms that could affect the regulation of MDR1 expression. The A genotype of all SNPs demonstrated both lowest MDR1 values and significantly decreased OS (P < 0.05) with a high probability of relapses (P < 0.01). These observations indicate that allelic variants of the MDR1 gene may influence therapy outcome by additional mechanisms, different from P-gp expression on acute myeloid leukemia blasts, possibly involving pharmacokinetic effects of P-gp.