Mesodermal Progenitor Cells Research Articles

Optimizing Nodal, Wnt and BMP signaling pathways for robust and efficient differentiation of human induced pluripotent stem cells to intermediate mesoderm cells.

Several differentiation protocols have enabled the generation of intermediate mesoderm (IM)-derived cells from human pluripotent stem cells (hPSC). However, the substantial variability between existing protocols for generating IM cells compromises their efficiency, reproducibility, and overall success, potentially hindering the utility of urogenital system organoids. Here, we examined the role of high levels of Nodal signaling and BMP activity, as well as WNT signaling in the specification of IM cells derived from a UCSD167i-99-1 human induced pluripotent stem cells (hiPSC) line. We demonstrate that precise modulation of WNT and BMP signaling significantly enhances IM differentiation efficiency. Treatment of hPSC with 3μM CHIR99021 induced TBXT+/MIXL1+ mesoderm progenitor (MP) cells after 48h of differentiation. Further treatment with a combination of 3μM CHIR99021 and 4ng/mL BMP4 resulted in the generation of OSR1+/GATA3+/PAX2+ IM cells within a subsequent 48h period. Molecular characterization of differentiated cells was confirmed through immunofluorescence staining and RT-qPCR. Hence, this study establishes a consistent and reproducible protocol for differentiating hiPSC into IM cells that faithfully recapitulates the molecular signatures of IM development. This protocol holds promise for improving the success of protocols designed to generate urogenital system organoids in vitro, with potential applications in regenerative medicine, drug discovery, and disease modeling.

HRAS-related epidermal nevus syndromes: Expansion of the spectrum with first branchial arch defects.

Epidermal nevus syndrome (ENS) comprises a heterogeneous group of neurocutaneous syndromes associated with the presence of epidermal nevi and variable extracutaneous manifestations. Postzygotic activating HRAS pathogenic variants were previously identified in nevus sebaceous (NS), keratinocytic epidermal nevus (KEN), and different ENS, including Schimmelpenning-Feuerstein-Mims and cutaneous-skeletal-hypophosphatasia syndrome (CSHS). Skeletal involvement in HRAS-related ENS ranges from localized bone dysplasia in association with KEN to fractures and limb deformities in CSHS. We describe the first association of HRAS-related ENS and auricular atresia, thereby expanding the disease spectrum with first branchial arch defects if affected by the mosaic variant. In addition, this report illustrates the first concurrent presence of verrucous EN, NS, and nevus comedonicus (NC), indicating the possibility of mosaic HRAS variation as an underlying cause of NC. Overall, this report extends the pleiotropy of conditions associated with mosaic pathogenic variants in HRAS affecting ectodermal and mesodermal progenitor cells.

Endothelial Differentiation of CCM1 Knockout iPSCs Triggers the Establishment of a Specific Gene Expression Signature.

Cerebral cavernous malformation (CCM) is a neurovascular disease that can lead to seizures and stroke-like symptoms. The familial form is caused by a heterozygous germline mutation in either the CCM1, CCM2, or CCM3 gene. While the importance of a second-hit mechanism in CCM development is well established, it is still unclear whether it immediately triggers CCM development or whether additional external factors are required. We here used RNA sequencing to study differential gene expression in CCM1 knockout induced pluripotent stem cells (CCM1-/- iPSCs), early mesoderm progenitor cells (eMPCs), and endothelial-like cells (ECs). Notably, CRISPR/Cas9-mediated inactivation of CCM1 led to hardly any gene expression differences in iPSCs and eMPCs. However, after differentiation into ECs, we found the significant deregulation of signaling pathways well known to be involved in CCM pathogenesis. These data suggest that a microenvironment of proangiogenic cytokines and growth factors can trigger the establishment of a characteristic gene expression signature upon CCM1 inactivation. Consequently, CCM1-/- precursor cells may exist that remain silent until entering the endothelial lineage. Collectively, not only downstream consequences of CCM1 ablation but also supporting factors must be addressed in CCM therapy development.

Foregut organ progenitors and their niche display distinct viscoelastic properties in vivo during early morphogenesis stages

Material properties of living matter play an important role for biological function and development. Yet, quantification of material properties of internal organs in vivo, without causing physiological damage, remains challenging. Here, we present a non-invasive approach based on modified optical tweezers for quantifying sub-cellular material properties deep inside living zebrafish embryos. Material properties of cells within the foregut region are quantified as deep as 150 µm into the biological tissue through measurements of the positions of an inert tracer. This yields an exponent, α, which characterizes the scaling behavior of the positional power spectra and the complex shear moduli. The measurements demonstrate differential mechanical properties: at the time when the developing organs undergo substantial displacements during morphogenesis, gut progenitors are more elastic (α = 0.57 ± 0.07) than the neighboring yolk (α = 0.73 ± 0.08), liver (α = 0.66 ± 0.06) and two mesodermal (α = 0.68 ± 0.06, α = 0.64 ± 0.06) progenitor cell populations. The higher elasticity of gut progenitors correlates with an increased cellular concentration of microtubules. The results infer a role of material properties during morphogenesis and the approach paves the way for quantitative material investigations in vivo of embryos, explants, or organoids.

3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR Tcells.

Unlimited generation of chimeric antigen receptor (CAR) Tcells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR Tcell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ Tcell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory Tcells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR Tcell characteristics. Expanded iPSC CD19-CAR Tcells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR Tcells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR Tcells also mediated potent antitumor activity invivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR Tcells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.

Nicotinamide promotes cardiomyocyte derivation and survival through kinase inhibition in human pluripotent stem cells

Nicotinamide, the amide form of Vitamin B3, is a common nutrient supplement that plays important role in human fetal development. Nicotinamide has been widely used in clinical treatments, including the treatment of diseases during pregnancy. However, its impacts during embryogenesis have not been fully understood. In this study, we show that nicotinamide plays multiplex roles in mesoderm differentiation of human embryonic stem cells (hESCs). Nicotinamide promotes cardiomyocyte fate from mesoderm progenitor cells, and suppresses the emergence of other cell types. Independent of its functions in PARP and Sirtuin pathways, nicotinamide modulates differentiation through kinase inhibition. A KINOMEscan assay identifies 14 novel nicotinamide targets among 468 kinase candidates. We demonstrate that nicotinamide promotes cardiomyocyte differentiation through p38 MAP kinase inhibition. Furthermore, we show that nicotinamide enhances cardiomyocyte survival as a Rho-associated protein kinase (ROCK) inhibitor. This study reveals nicotinamide as a pleiotropic molecule that promotes the derivation and survival of cardiomyocytes, and it could become a useful tool for cardiomyocyte production for regenerative medicine. It also provides a theoretical foundation for physicians when nicotinamide is considered for treatments for pregnant women.

Abstract 11721: FOXK1 is an Essential Transcriptional and Epigenetic Regulator of Cardiovascular Development

Background: Congenital heart disease (CHD) is the most common genetic birth defect and has considerable morbidity and mortality. Therefore, it is essential to define the mechanisms governing the specification and differentiation of mesodermal and cardiac progenitors to develop targeted therapies for CHD. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics, myogenic stem cell proliferation and tumorigenesis. During development, FOXK1 expression is restricted to mesodermal progenitors, somites and the heart. The role for FOXK1 in the developing heart is unknown and warrants investigation. In the present study, we describe a novel role for FOXK1 as an essential regulator of cardiovascular development. Approach and Results: We used a mouse embryoid body (EB) system to differentiate WT and Foxk1 null ESCs into mesodermal and cardiac progenitor cells. Flow cytometry analysis for FLK1+PDGFRa+ showed that in the absence of FOXK1, the cardiac lineage was significantly affected. Bulk RNAseq analysis of D3 and D5 EBs showed a significant induction of the cardiac molecular program in WT EBs compared to the Foxk1 null EBs. Since forkhead transcription factors have been shown to be important epigenetic regulators (i.e. pioneer factors), we performed ATACseq of D3 and D5 EBs, which showed that the chromatin landscape nearby known important regulators of cardiogenesis (Isl1, Gata4, Hand1, Hand2, etc.) was significantly relaxed in WT EBs compared to Foxk1 null EBs. Furthermore, we demonstrated that in the absence of Foxk1 , cardiac differentiation was markedly impaired by assaying for cTnT expression and cardiac contractility which was essentially absent in Foxk1 null EBs compared to WT EBs at D10 of differentiation. Additionally, using transcriptional, Co-IP and pulldown assays we demonstrated that FOXK1 is an important regulator of Notch and Wnt/β-catenin signaling pathways during cardiogenesis. These results were further supported as the Foxk1 null embryo was lethal at E9.5 and had perturbed heart development. Conclusions: These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development using EB assays and gene disruption technologies.

Differentiating Human Pluripotent Stem Cells to Vascular Endothelial Cells for Regenerative Medicine, Tissue Engineering, and Disease Modeling.

Vasculature plays a vital role in human biology as blood vessels transport nutrients and oxygen throughout the body. Endothelial cells (ECs), specifically, are key as they maintain barrier functions between the circulating blood and the surrounding tissues. ECs derived from human pluripotent stem cells (hPSCs) are utilized to study vascular development and disease mechanisms within in vitro models. Additionally, ECs derived from induced pluripotent stem cells (iPSCs) hold great promise for advancing personalized medicine, cell therapies, and tissue-engineered constructs by creating patient-specific cell populations. Here, we describe a xeno-free, serum-free differentiation protocol for deriving ECs from hPSCs. In brief, mesoderm progenitor cells are derived via WNT pathway activation. Following this, EC maturation is achieved with exogenous vascular endothelial growth factor A (VEGFA) and basic fibroblast growth factor 2 (bFGF2). We have characterized these cells as expressing mature EC markers and have illustrated their functionality in vitro.

Human iPSC-derived mesodermal progenitor cells preserve their vasculogenesis potential after extrusion and form hierarchically organized blood vessels

Post-fabrication formation of a proper vasculature remains an unresolved challenge in bioprinting. Established strategies focus on the supply of the fabricated structure with nutrients and oxygen and either rely on the mere formation of a channel system using fugitive inks or additionally use mature endothelial cells and/or peri-endothelial cells such as smooth muscle cells for the formation of blood vessels in vitro. Functional vessels, however, exhibit a hierarchical organization and multilayered wall structure that is important for their function. Human induced pluripotent stem cell-derived mesodermal progenitor cells (hiMPCs) have been shown to possess the capacity to form blood vessels in vitro, but have so far not been assessed for their applicability in bioprinting processes. Here, we demonstrate that hiMPCs, after formulation into an alginate/collagen type I bioink and subsequent extrusion, retain their ability to give rise to the formation of complex vessels that display a hierarchical network in a process that mimics the embryonic steps of vessel formation during vasculogenesis. Histological evaluations at different time points of extrusion revealed the initial formation of spheres, followed by lumen formation and further structural maturation as evidenced by building a multilayered vessel wall and a vascular network. These findings are supported by immunostainings for endothelial and peri-endothelial cell markers as well as electron microscopic analyses at the ultrastructural level. Moreover, endothelial cells in capillary-like vessel structures deposited a basement membrane-like matrix at the basal side between the vessel wall and the alginate-collagen matrix. After transplantation of the printed constructs into the chicken chorioallantoic membrane (CAM) the printed vessels connected to the CAM blood vessels and get perfused in vivo. These results evidence the applicability and great potential of hiMPCs for the bioprinting of vascular structures mimicking the basic morphogenetic steps of de novo vessel formation during embryogenesis.

SCA-1/Ly6A Mesodermal Skeletal Progenitor Subpopulations Reveal Differential Commitment of Early Limb Bud Cells

At early developmental stages, limb bud mesodermal undifferentiated cells are morphologically indistinguishable. Although the identification of several mesodermal skeletal progenitor cell populations has been recognized, in advanced stages of limb development here we identified and characterized the differentiation hierarchy of two new early limb bud subpopulations of skeletal progenitors defined by the differential expression of the SCA-1 marker. Based on tissue localization of the mesenchymal stromal cell-associated markers (MSC-am) CD29, Sca-1, CD44, CD105, CD90, and CD73, we identified, by multiparametric analysis, the presence of cell subpopulations in the limb bud capable of responding to inductive signals differentially, namely, sSca+ and sSca– cells. In concordance with its gene expression profile, cell cultures of the sSca+ subpopulation showed higher osteogenic but lower chondrogenic capacity than those of sSca–. Interestingly, under high-density conditions, fibroblast-like cells in the sSca+ subpopulation were abundant. Gain-of-function employing micromass cultures and the recombinant limb assay showed that SCA-1 expression promoted tenogenic differentiation, whereas chondrogenesis is delayed. This model represents a system to determine cell differentiation and morphogenesis of different cell subpopulations in similar conditions like in vivo. Our results suggest that the limb bud is composed of a heterogeneous population of progenitors that respond differently to local differentiation inductive signals in the early stages of development, where SCA-1 expression may play a permissive role during cell fate.

Poly(vinyl alcohol)/Gelatin Scaffolds Allow Regeneration of Nasal Tissues

Need for regeneration and repair of nasal tissues occurs as a consequence of several pathologies affecting the nose, including, but not limited to infective diseases, traumas and tumor resections. A platform for nasal tissue regeneration was set up using poly(vinyl alcohol)/gelatin sponges with 20%–30% (w/w) gelatin content to be used as scaffolds, for their intrinsic hydrophilic, cell adhesive and shape recovery properties. We propose mesodermal progenitor cells (MPCs) isolated from the bone marrow as a unique stem cell source for obtaining different connective tissues of the nose, including vascular tissue. Finally, epithelial cell immune response to these scaffolds was assessed in vitro in an environment containing inflammatory molecules. The results showed that mesenchymal stromal cells (MSCs) deriving from MPCs could be used to differentiate into cartilage and fibrous tissue; whereas, in combination with endothelial cells still deriving from MPCs, into pre-vascularized bone. Finally, the scaffold did not significantly alter the epithelial cell response to inflammatory insults derived from interaction with bacterial molecules.

The CXCR4/SDF-1 Axis in the Development of Facial Expression and Non-somitic Neck Muscles.

Trunk and head muscles originate from distinct embryonic regions: while the trunk muscles derive from the paraxial mesoderm that becomes segmented into somites, the majority of head muscles develops from the unsegmented cranial paraxial mesoderm. Differences in the molecular control of trunk versus head and neck muscles have been discovered about 25 years ago; interestingly, differences in satellite cell subpopulations were also described more recently. Specifically, the satellite cells of the facial expression muscles share properties with heart muscle. In adult vertebrates, neck muscles span the transition zone between head and trunk. Mastication and facial expression muscles derive from the mesodermal progenitor cells that are located in the first and second branchial arches, respectively. The cucullaris muscle (non-somitic neck muscle) originates from the posterior-most branchial arches. Like other subclasses within the chemokines and chemokine receptors, CXCR4 and SDF-1 play essential roles in the migration of cells within a number of various tissues during development. CXCR4 as receptor together with its ligand SDF-1 have mainly been described to regulate the migration of the trunk muscle progenitor cells. This review first underlines our recent understanding of the development of the facial expression (second arch-derived) muscles, focusing on new insights into the migration event and how this embryonic process is different from the development of mastication (first arch-derived) muscles. Other muscles associated with the head, such as non-somitic neck muscles derived from muscle progenitor cells located in the posterior branchial arches, are also in the focus of this review. Implications on human muscle dystrophies affecting the muscles of face and neck are also discussed.

SWI/SNF Component BAF250a Coordinates OCT4 and WNT Signaling Pathway to Control Cardiac Lineage Differentiation.

Dissecting epigenetic mechanisms controlling early cardiac differentiation will provide insights into heart regeneration and heart disease treatment. SWI/SNF complexes remodel nucleosomes to regulate gene expression and play a key role in organogenesis. Here, we reported a unique function of BAF250a in regulating the physical interaction of OCT4 and β-CATENIN during cardiac lineage differentiation from human ESCs. BAF250a deletion greatly reduced the physical interaction between OCT4 and β-CATENIN but did not alter the expression of β-CATENIN and OCT4 in the mesodermal progenitor cells. BAF250a ablation led to decreased recruitment of OCT4 and β-CATENIN at promoters of key mesodermal lineage genes, such as MESP1 and EOMES. Subsequently, the expression of lineage-specific genes was downregulated, whereas the expression of pluripotent genes was upregulated. In parallel, BAF250a ablation also altered recruitments of OCT4 and β-CATENIN to the promoter of CCND2 and CCND3, two key genes for S phase entry during cell cycle. Consequently, BAF250a deletion led to prolonged S phase in Mesp1+ cardiac progenitor cells, which in turn inhibited efficient differentiation of Mesp1+ to Isl1+ cells. Furthermore, BAF250a deletion abolished the interaction of OCT4 and BRG1 in mesoderm, suggesting that BAF250a is the key component in SWI/SNF complex that determines the interaction of Oct4/β-catenin in mesoderm. In contrast, we found that BAF250a did not regulate the OCT4/β-CATENIN interaction during neuroectoderm differentiation. Altogether, our results suggest that BAF250a specifically controls proper cardiac mesoderm differentiation by reorganizing the binding of OCT4/β-CATENIN and regulates both key lineage differentiation genes and cell cycle genes that coincided in response to WNT/β-CATENIN signal.

In ovo technique for cell injection in the CPM followed by bead implantation in the BA2 of chicken embryos

The major advantage of chicken embryos model is their accessibility for microsurgical manipulations and the dissection of tissues for ex vivo explant culture. Branchial arches are embryonic structure located next to the top of developing heart. Each arch is made of surface ectoderm, endoderm, myogenic mesoderm cells and cranial neural crest cells. The myogenic mesoderm originates from cranial paraxial mesoderm (CPM), which is transiently migrated to branchial arches (BAs). The first branchial arch (BA1) mesoderm contributes to formation of mastication muscles. The second branchial arch (BA2) mesoderm gives rise to facial expression muscles. This article focuses on cell injection in the CPM and bead implantation (gain of function approaches) in the BA2. In order to follow the migration of mesoderm progenitor cells from CPM to BA2, we injected quail cells in the CPM of stage HH10-11 embryos, followed by implantation of SDF-1 bead at stage HH15-16. Later the attraction of quail cells (CXCR4+) towards the SDF-1 source has been observed, using whole-mount immunostaining of a specific quail antibody (QCPN) at stage HH19-22.•Our method, which involves bead implantation followed by quail cell injection, provides useful tools for tracing migratory mesodermal cells in vivo.•The proposed method does not require any commercial kits and can be used for various developmental process.•It does not employ any complicated methods such as genetically engineered permanent cell labeling, multiplicity of fluorescent markers or clonal analysis.

Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells

Organoids derived from human pluripotent stem cells are interesting models to study mechanisms of morphogenesis and promising platforms for disease modeling and drug screening. However, they mostly remain incomplete as they lack stroma, tissue resident immune cells and in particular vasculature, which create important niches during development and disease. We propose, that the directed incorporation of mesodermal progenitor cells (MPCs) into organoids will overcome the aforementioned limitations. In order to demonstrate the feasibility of the method, we generated complex human tumor as well as neural organoids. We show that the formed blood vessels display a hierarchic organization and mural cells are assembled into the vessel wall. Moreover, we demonstrate a typical blood vessel ultrastructure including endothelial cell-cell junctions, a basement membrane as well as luminal caveolae and microvesicles. We observe a high plasticity in the endothelial network, which expands, while the organoids grow and is responsive to anti-angiogenic compounds and pro-angiogenic conditions such as hypoxia. We show that vessels within tumor organoids connect to host vessels following transplantation. Remarkably, MPCs also deliver Iba1+ cells that infiltrate the neural tissue in a microglia-like manner.

Hedgehog-related genes regulate reactivation of quiescent neural progenitors in Caenorhabditis elegans

The animal body contains various types of stem and progenitor cells. These undifferentiated cells coordinate the balance between quiescence and proliferation with dynamics of various physiological conditions such as the developmental stage, food availability, and injury. Although regulation of such coordination plays a critical role in maintaining tissue homeostasis, controlling the growth rate and regeneration, much of its mechanism remains elusive. Newly hatched Caenorhabditis elegans larvae possess quiescent stem and progenitor cells in several tissues, and these cells are reactivated by the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway only when sufficient food is supplied.Maintenance of the quiescence of neuronal and mesodermal progenitor cells requires microRNA (miRNA), miR-235, which is upregulated under the starvation. On the other hand, feeding ample food downregulates the miRNA via the activity of the IIS pathway. As miR-235 in the hypodermis can non-autonomously regulate quiescence of neuronal and mesodermal progenitor cells, a cell-cell signaling pathway has been hypothesized to act downstream of the miRNA.Here, we provide evidence that two hedgehog-related (hh-r) genes, grl-5 and grl-7, are targets of miR-235 that promote reactivation of quiescent neuroblasts. These grl genes possess an miR-235 binding site on 3ʹUTRs of their transcripts, and are upregulated in starved mir-235 mutant larvae. grl-5 and grl-7 promoters can continuously drive the expression of GFP-pest reporter protein in the hypodermis under the fed condition. However, expression of these reporters is strikingly downregulated under the starvation condition after hatching. We found that miR-235 can repress expression of reporter genes via the predicted miR-235 binding sites on the grl-5 and grl-7 3ʹUTRs. Furthermore, activity of grl-5 and grl-7 genes are required for reactivation of neural progenitor cells in starved mir-235 mutant larvae. These findings suggest that the IIS pathway-miR-235 signaling in the hypodermis non-autonomously regulates quiescence of neural progenitor cells, partly via grl-5 and grl-7.

Tbx6 induces cardiomyocyte proliferation in postnatal and adult mouse hearts

Cardiovascular disease is a leading cause of death worldwide. Mammalian cardiomyocytes (CMs) proliferate during embryonic development, whereas they largely lose their regenerative capacity after birth. Defined factors expressed in cardiac progenitors or embryonic CMs may activate the cell cycle and induce CM proliferation in postnatal and adult hearts. Here, we report that the overexpression of Tbx6, enriched in the cardiac mesoderm (progenitor cells), induces CM proliferation in postnatal and adult mouse hearts. By screening 24 factors enriched in cardiac progenitors or embryonic CMs, we found that only Tbx6 could induce CM proliferation in primary cultured postnatal rat CMs. Intriguingly, it did not induce the proliferation of cardiac fibroblasts. We next generated a recombinant adeno-associated virus serotype 9 vector encoding Tbx6 (AAV9-Tbx6) for transduction into mouse CMs in vivo. The subcutaneous injection of AAV9-Tbx6 into neonatal mice induced CM proliferation in postnatal and adult mouse hearts. Mechanistically, Tbx6 overexpression upregulated multiple cell cycle activators including Aurkb, Mki67, Ccna1, and Ccnb2 and suppressed the tumor suppressor Rb1. Thus, Tbx6 promotes CM proliferation in postnatal and adult mouse hearts by modifying the expression of cell cycle regulators.

GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells

BackgroundHuman mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines.MethodsIn the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium).ResultsThe results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum.ConclusionsThe findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine.Graphical abstract

Identification of the miRNAome of early mesoderm progenitor cells and cardiomyocytes derived from human pluripotent stem cells

MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression related to many cellular functions. We performed a small-RNAseq analysis of cardiac differentiation from pluripotent stem cells. Our analyses identified some new aspects about microRNA expression in this differentiation process. First, we described a dynamic expression profile of microRNAs where some of them are clustered according to their expression level. Second, we described the extensive network of isomiRs and ADAR modifications. Third, we identified the microRNAs families and clusters involved in the establishment of cardiac lineage and define the mirRNAome based on these groups. Finally, we were able to determine a more accurate miRNAome associated with cardiomyocytes by comparing the expressed microRNAs with other mature cells. MicroRNAs exert their effect in a complex and interconnected way, making necessary a global analysis to better understand their role. Our data expands the knowledge of microRNAs and their implications in cardiomyogenesis.

Identification of Adult Mesodermal Progenitor Cells and Hierarchy in Atherosclerotic Vascular Calcification

The nature of calcifying progenitor cells remains elusive. In this study, we investigated the developmental hierarchy and dynamics of progenitor cells. In vitro and in vivo reconstitution assays demonstrated that Sca-1+/PDGFRα- cells in the bone marrow (BM) are the ancestors of Sca-1+/PDGFRα+ cells. Cells of CD29 + Sca-1+/PDGFRα- lineage in the BM showed both hematopoietic potential with osteoclastic differentiation ability as well as mesenchymal stem cell-like properties with osteoblastic differentiation potential. Clonally-isolated BM-derived artery-infiltrated Sca-1+/PDGFRα- cells maintained osteoblastic/osteoclastic bipotency but lost hematopoietic activity. In hypercholesterolemic apolipoprotein-E-deficient (Apoe-/-) mice, the mobilization from BM to peripheral circulation, followed by migration into atherosclerotic plaques of Sca-1+/PDGFRα- cells, but not Sca-1+/PDGFRα+ cells, were significantly decreased, and Interleukin-1β (IL-1β) and Interleukin-5 (IL-5) mediated this response. Here, we demonstrated that Sca-1+/PDGFRα- cells are mesodermal progenitor cells in adults, and the dynamics of progenitor cells were regulated by atherosclerosis-related humoral factors. These results may contribute to better understanding of vascular homeostasis and assist in the development of novel therapies for atherosclerosis. Stem Cells 2018;36:1075-1096.