Mesodermal Lineages Research Articles

ETV2 and VEZF1 interaction and regulation of the hematoendothelial lineage during embryogenesis.

Ets variant 2 (Etv2), a member of the Ets factor family, has an essential role in the formation of endothelial and hematopoietic cell lineages during embryonic development. The functional role of ETS transcription factors is, in part, dependent on the interacting proteins. There are relatively few studies exploring the coordinated interplay between ETV2 and its interacting proteins that regulate mesodermal lineage determination. In order to identify novel ETV2 interacting partners, a yeast two-hybrid analysis was performed and the C2H2 zinc finger transcription factor VEZF1 (vascular endothelial zinc finger 1) was identified as a binding factor, which was specifically expressed within the endothelium during vascular development. To confirm this interaction, co-immunoprecipitation and GST pull down assays demonstrated the direct interaction between ETV2 and VEZF1. During embryoid body differentiation, Etv2 achieved its peak expression at day 3.0 followed by rapid downregulation, on the other hand Vezf1 expression increased through day 6 of EB differentiation. We have previously shown that ETV2 potently activated Flt1 gene transcription. Using a Flt1 promoter-luciferase reporter assay, we demonstrated that VEZF1 co-activated the Flt1 promoter. Electrophoretic mobility shift assay and Chromatin immunoprecipitation established VEZF1 binding to the Flt1 promoter. Vezf1 knockout embryonic stem cells had downregulation of hematoendothelial marker genes when undergoing embryoid body mediated mesodermal differentiation whereas overexpression of VEZF1 induced the expression of hematoendothelial genes during differentiation. These current studies provide insight into the co-regulation of the hemato-endothelial lineage development via a co-operative interaction between ETV2 and VEZF1.

The Proteomes of Oral Cells Change during Co-Cultivation with Aggregatibacter actinomycetemcomitans and Eikenella corrodens

Changes in the proteome of oral cells during periodontitis have rarely been investigated. This lack of information is partially attributed to the lack of human cell lines derived from the oral cavity for in vitro research. The objective of the present study was to create cell lines from relevant oral tissues and compare protein expression in cells cultured alone and in cells co-cultivated with periodontitis-associated bacterial strains. We established human cell lines of gingival keratinocytes, osteoblastic lineage cells from the alveolar bone, periodontal ligament fibroblasts, and cementum cells. Using state-of-the-art label-free mass spectrometry, we investigated changes in the proteomes of these cells after co-cultivation with Aggregatibacter actinomycetemcomitans and Eikenella corrodens for 48 h. Gingival keratinocytes, representing ectodermal cells, exhibited decreased expression of specific keratins, basement membrane components, and cell-cell contact proteins after cultivation with the bacterial strains. Mesodermal lineage cells generally exhibited similar proteomes after co-cultivation with bacteria; in particular, collagens and integrins were expressed at higher levels. The results of the present study will help us elucidate the cellular mechanisms of periodontitis. Although co-cultivation with two periodontitis-associated bacterial strains significantly altered the proteomes of oral cells, future research is needed to examine the effects of complex biofilms mimicking in vivo conditions.

Protein Kinase C Modulation Determines the Mesoderm/Extraembryonic Fate Under BMP4 Induction From Human Pluripotent Stem Cells.

The interplay among mitogenic signaling pathways is crucial for proper embryogenesis. These pathways collaboratively act through intracellular master regulators to determine specific cell fates. Identifying the master regulators is critical to understanding embryogenesis and to developing new applications of pluripotent stem cells. In this report, we demonstrate protein kinase C (PKC) as an intrinsic master switch between embryonic and extraembryonic cell fates in the differentiation of human pluripotent stem cells (hPSCs). PKCs are essential to induce the extraembryonic lineage downstream of BMP4 and other mitogenic modulators. PKC-alpha (PKCα) suppresses BMP4-induced mesoderm differentiation, and PKC-delta (PKCδ) is required for trophoblast cell fate. PKC activation overrides mesoderm induction conditions and leads to extraembryonic fate. In contrast, PKC inhibition leads to β-catenin (CTNNB1) activation, switching cell fate from trophoblast to mesoderm lineages. This study establishes PKC as a signaling boundary directing the segregation of extraembryonic and embryonic lineages. The manipulation of intrinsic PKC activity could greatly enhance cell differentiation under mitogenic regulation in stem cell applications.

A three-component model of the spinal nerve ramification: Bringing together the human gross anatomy and modern Embryology.

Due to its long history, the study of human gross anatomy has not adequately incorporated modern embryological findings; consequently, the current understanding has often been incompatible with recent discoveries from molecular studies. Notably, the traditional epaxial and hypaxial muscle distinction, and their corresponding innervation by the dorsal and ventral rami of the spinal nerve, do not correspond to the primaxial and abaxial muscle distinction, defined by the mesodermal lineages of target tissues. To resolve the disagreement between adult anatomy and embryology, we here propose a novel hypothetical model of spinal nerve ramification. Our model is based on the previously unknown developmental process of the intercostal nerves. Observations of these nerves in the mouse embryos revealed that the intercostal nerves initially had superficial and deep ventral branches, which is contrary to the general perception of a single ventral branch. The initial dual innervation pattern later changes into an adult-like single branch pattern following the retraction of the superficial branch. The modified intercostal nerves consist of the canonical ventral branches and novel branches that run on the muscular surface of the thorax, which sprout from the lateral cutaneous branches. We formulated the embryonic branching pattern into the hypothetical ramification model of the human spinal nerve so that the branching pattern is compatible with the developmental context of the target muscles. In our model, every spinal nerve consists of three components: (1) segmental branches that innervate the primaxial muscles, including the dorsal rami, and short branches and long superficial anterior branches from the ventral rami; (2) plexus-forming intramural branches, the serial homolog of the canonical intercostal nerves, which innervate the abaxial portion of the body wall; and (3) plexus-forming extramural branches, the series of novel branches located outside of the body wall, which innervate the girdle and limb muscles. The selective elaboration or deletion of each component successfully explains the reasoning for the standard morphology and variability of the spinal nerve. Therefore, our model brings a novel understanding of spinal nerve development and valuable information for basic and clinical sciences regarding the diverse branching patterns of the spinal nerve.

Potential Therapy Intrathracheal Uses Umbilical Cord Blood Mesencimal Stem Cell Derivatives As The Latest Strategy For The Treatment Of Dysplasia Broncopulmonary In Premature Infants In Indonesia : A Literature Review

Indonesia is the 5th country with the highest premature birth rate in the world. Multifactorial complications in premature babies whose lungs are not yet fully developed in the form of respiratory distress syndrome, one of which is bronchopulmonary dysplasia, can hinder their growth and development in the future. Bronchopulmonary dysplasia (Bronchopulmonary dysplasia/BPD) is one of the most common causes of morbidity in premature infants. Globally, the incidence of BPD varies widely from 10% -89%. Utilization of stem cells or stem cells has become one of the futuristic approaches in treating diseases related to tissue engineering. The study found that Mesenchymal Stem Cells (MSC/Mesenchymal Stem Cells) which are multipotent and have the ability to differentiate into cells derived from the mesoderm lineage, can be a renewable step in repairing tissues in the lungs, BPD is no exception. Compared to adult stem cells, the use of embryonic stem cells, especially Mesenchymal Stem Cells (MSC) from umbilical cord blood, has the potential to self-renewal and differentiate into several types of cells in an unlimited way. MSC from cord blood has advantages including, 1) easy collection process, 2) rapid proliferation, 3) fast processing at a relatively cheap price compared to other sources, 4) minimal risk of disease transmission and rejection, 5) and safer for mother and child. MSC transplantation has potential as a therapy for BPD by acting as a “paracrine factory” that can detect and respond to cues in the microenvironment and the site of injury. However, the potential of MSC in BPD still needs to be explored further considering its enormous benefits. There needs to be holistic collaboration between health workers and researchers so that this method can be realized. Further research on side effects, therapeutic doses, and long-term effects of therapy is expected to complement the challenges that are still present today.

Effect of stingless bee (Hymenoptera: Apidae: Apinae: Meliponini) propolis on the proliferation of human stem cells

The effect of stingless bee propolis on the proliferation and differentiation of human stem cells is reported for the first time. Human pluripotent stem cells (hPSCs) treated with the propolis extracted from Lisotrigona sp, Tetragonula calophyllae and T. travancorica (Hymenoptera: Apidae: Apinae: Meliponini) displayed a remarkable difference in their morphology. Gene expression analysis revealed pluripotency markers OCT4 and NANOG to be down-regulated upon treatment with propolis, which confirmed early differentiation of hPSCs. Further investigation on the gene expression of early differentiation markers revealed that propolis supports mesendoderm differentiation, which is a novel finding. The propolis obtained from stingless bees Tetragonula spp. probably has more therapeutic value in terms of its effect on hPSCs viz., more tendency of the cells to differentiate into mesoderm and endoderm lineages, compared to the propolis obtained from Lisotrigona sp.

Declined adipogenic potential of senescent MSCs due to shift in insulin signaling and altered exosome cargo.

Multipotent mesenchymal stromal cells (MSCs) maintain cellular homeostasis and regulate tissue renewal and repair both by differentiating into mesodermal lineage, e.g., adipocytes, or managing the functions of differentiated cells. Insulin is a key physiological inducer of MSC differentiation into adipocytes, and disturbances in MSC insulin sensitivity could negatively affect adipose tissue renewal. During aging, regulation and renewal of adipose tissue cells may be disrupted due to the altered insulin signaling and differentiation potential of senescent MSCs, promoting the development of serious metabolic diseases, including metabolic syndrome and obesity. However, the potential mechanisms mediating the dysfunction of adipose-derived senescent MSC remains unclear. We explored whether aging could affect the adipogenic potential of human adipose tissue-derived MSCs regulated by insulin. Age-associated senescent MSCs (isolated from donors older than 65years) and MSCs in replicative senescence (long-term culture) were treated by insulin to induce adipogenic differentiation, and the efficiency of the process was compared to MSCs from young donors. Insulin-dependent signaling pathways were explored in these cells. We also analyzed the involvement of extracellular vesicles secreted by MSCs (MSC-EVs) into the regulation of adipogenic differentiation and insulin signaling of control and senescent cells. Also the microRNA profiles of MSC-EVs from aged and young donors were compared using targeted PCR arrays. Both replicatively and chronologically senescent MSCs showed a noticeably decreased adipogenic potential. This was associated with insulin resistance of MSCs from aged donors caused by the increase in the basal level of activation of crucial insulin-dependent intracellular effectors ERK1/2 and Akt. To assess the impact of the paracrine cross-talk of MSCs, we analyzed microRNAs profile differences in MSC-EVs and revealed that senescent MSCs produced EVs with increased content of miRNAs targeting components of insulin-dependent signaling cascade PTEN, MAPK1, GAREM1 and some other targets. We also confirmed these data by differentiation of control MSCs in the presence of EVs from senescent cells and vice versa. Thus, aging attenuated the adipogenic potential of MSCs due to autocrine or paracrine-dependent induction of insulin resistance associated with the specific changes in MSC-EV cargo.

Chondrogenic Differentiation of Human-Induced Pluripotent Stem Cells.

The generation of large quantities of genetically defined human chondrocytes remains a critical step for the development of tissue engineering strategies for cartilage regeneration and high-throughput drug screening. This protocol describes chondrogenic differentiation of human-induced pluripotent stem cells (hiPSCs), which can undergo genetic modification and the capacity for extensive cell expansion. The hiPSCs are differentiated in a stepwise manner in monolayer through the mesodermal lineage for 12days using defined growth factors and small molecules. This is followed by 28days of chondrogenic differentiation in a 3D pellet culture system using transforming growth factor beta 3 and specific compounds to inhibit off-target differentiation. The 6-week protocol results in hiPSC-derived cartilaginous tissue that can be characterized by histology, immunohistochemistry, and gene expression or enzymatically digested to isolate chondrocyte-like cells. Investigators can use this protocol for experiments including genetic engineering, in vitro disease modeling, or tissue engineering.

Mechano-regulation of Wnt/β-Catenin signaling to control paraxial versus lateral mesoderm lineage bifurcation.

Mechanical cues are involved in many biological processes, including embryonic development and patterning. For example, external mechanical forces (shear stress), lateral cell-cell interactions, and mechanical properties (stiffness and composition) of the extracellular matrix are thought to modulate Wnt signaling, which is a highly conserved pathway involved in regulating stem cell renewal, proliferation, and differentiation. In this work, we employed a customized higher-throughput shear stress induction device for the controlled application of mechanical stress to study the effects of shear stress on the differentiation of human induced pluripotent stem cells (hiPSCs) toward the three germ layers. We found that mechanical stress alters lineage commitment during ectoderm and mesoderm differentiation. We show that this effect correlates with reduced Wnt signaling, evaluated in terms of the promoter activity of an established TCF3-responsive promoter. Whole transcriptome sequencing and pathway enrichment analysis of the differentially expressed genes between hiPSC-derived mesoderm cells differentiated in the presence or absence of piston-induced shear stress confirmed that Wnt/β-catenin signaling is among the most affected developmental pathways. Furthermore, our results suggest that suitably programmed shear stress application could be used to selectively promote differentiation of hiPSCs to either lateral or paraxial mesoderm in commercially available media.

IPSC-neural crest derived cells embedded in 3D printable bio-ink promote cranial bone defect repair

Cranial bone loss presents a major clinical challenge and new regenerative approaches to address craniofacial reconstruction are in great demand. Induced pluripotent stem cell (iPSC) differentiation is a powerful tool to generate mesenchymal stromal cells (MSCs). Prior research demonstrated the potential of bone marrow-derived MSCs (BM-MSCs) and iPSC-derived mesenchymal progenitor cells via the neural crest (NCC-MPCs) or mesodermal lineages (iMSCs) to be promising cell source for bone regeneration. Overexpression of human recombinant bone morphogenetic protein (BMP)6 efficiently stimulates bone formation. The study aimed to evaluate the potential of iPSC-derived cells via neural crest or mesoderm overexpressing BMP6 and embedded in 3D printable bio-ink to generate viable bone graft alternatives for cranial reconstruction. Cell viability, osteogenic potential of cells, and bio-ink (Ink-Bone or GelXa) combinations were investigated in vitro using bioluminescent imaging. The osteogenic potential of bio-ink-cell constructs were evaluated in osteogenic media or nucleofected with BMP6 using qRT-PCR and in vitro μCT. For in vivo testing, two 2 mm circular defects were created in the frontal and parietal bones of NOD/SCID mice and treated with Ink-Bone, Ink-Bone + BM-MSC-BMP6, Ink-Bone + iMSC-BMP6, Ink-Bone + iNCC-MPC-BMP6, or left untreated. For follow-up, µCT was performed at weeks 0, 4, and 8 weeks. At the time of sacrifice (week 8), histological and immunofluorescent analyses were performed. Both bio-inks supported cell survival and promoted osteogenic differentiation of iNCC-MPCs and BM-MSCs in vitro. At 4 weeks, cell viability of both BM-MSCs and iNCC-MPCs were increased in Ink-Bone compared to GelXA. The combination of Ink-Bone with iNCC-MPC-BMP6 resulted in an increased bone volume in the frontal bone compared to the other groups at 4 weeks post-surgery. At 8 weeks, both iNCC-MPC-BMP6 and iMSC-MSC-BMP6 resulted in an increased bone volume and partial bone bridging between the implant and host bone compared to the other groups. The results of this study show the potential of NCC-MPC-incorporated bio-ink to regenerate frontal cranial defects. Therefore, this bio-ink-cell combination should be further investigated for its therapeutic potential in large animal models with larger cranial defects, allowing for 3D printing of the cell-incorporated material.

Platr4 is an early embryonic lncRNA that exerts its function downstream on cardiogenic mesodermal lineage commitment.

The mammalian genome encodes thousands of long non-coding RNAs (lncRNAs), many of which are developmentally regulated and differentially expressed across tissues, suggesting their potential roles in cellular differentiation. Despite this expression pattern, little is known about how lncRNAs influence lineage commitment at the molecular level. Here, we demonstrate that perturbation of an embryonic stem cell/early embryonic lncRNA, pluripotency-associated transcript 4 (Platr4), directly influences the specification of cardiac-mesoderm-lineage differentiation. We show that Platr4 acts as a molecular scaffold or chaperone interacting with the Hippo-signaling pathway molecules Yap and Tead4 to regulate the expression of a downstream target gene, Ctgf, which is crucial to the cardiac-lineage program. Importantly, Platr4 knockout mice exhibit myocardial atrophy and valve mucinous degeneration, which are both associated with reduced cardiac output and sudden heart failure. Together, our findings provide evidence that Platr4 is required in cardiac-lineage specification and adult heart function in mice.

The centrosomal protein 83 (CEP83) regulates human pluripotent stem cell differentiation toward the kidney lineage.

During embryonic development, the mesoderm undergoes patterning into diverse lineages including axial, paraxial, and lateral plate mesoderm (LPM). Within the LPM, the so-called intermediate mesoderm (IM) forms kidney and urogenital tract progenitor cells, while the remaining LPM forms cardiovascular, hematopoietic, mesothelial, and additional progenitor cells. The signals that regulate these early lineage decisions are incompletely understood. Here, we found that the centrosomal protein 83 (CEP83), a centriolar component necessary for primary cilia formation and mutated in pediatric kidney disease, influences the differentiation of human-induced pluripotent stem cells (hiPSCs) toward IM. We induced inactivating deletions of <i>CEP83</i> in hiPSCs and applied a 7-day in vitro protocol of IM kidney progenitor differentiation, based on timed application of WNT and FGF agonists. We characterized induced mesodermal cell populations using single-cell and bulk transcriptomics and tested their ability to form kidney structures in subsequent organoid culture. While hiPSCs with homozygous <i>CEP83</i> inactivation were normal regarding morphology and transcriptome, their induced differentiation into IM progenitor cells was perturbed. Mesodermal cells induced after 7 days of monolayer culture of <i>CEP83</i>-deficient hiPCS exhibited absent or elongated primary cilia, displayed decreased expression of critical IM genes (<i>PAX8</i>, <i>EYA1</i>, <i>HOXB7</i>), and an aberrant induction of LPM markers (e.g. <i>FOXF1</i>, <i>FOXF2</i>, <i>FENDRR</i>, <i>HAND1</i>, <i>HAND2</i>). Upon subsequent organoid culture, wildtype cells differentiated to form kidney tubules and glomerular-like structures, whereas <i>CEP83</i>-deficient cells failed to generate kidney cell types, instead upregulating cardiomyocyte, vascular, and more general LPM progenitor markers. Our data suggest that <i>CEP83</i> regulates the balance of IM and LPM formation from human pluripotent stem cells, identifying a potential link between centriolar or ciliary function and mesodermal lineage induction.

P125 Histoplasma capsulatum modulates the immune response exerted by mesenchymal stromal cells

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM BackgroundMesenchymal stromal cells (MSCs) have become a tool not only for tissue regeneration but also for the treatment of inflammatory diseases. Several studies have demonstrated the therapeutic potential of MSCs for the treatment of noninfectious inflammatory diseases; however, they appear to play a dual role in infectious diseases. Histoplasmosis is a systemic mycosis caused by Histoplasma spp., which occurs mainly in immunosuppressed individuals; this mycosis can present a severe clinical picture with dissemination to various organs and is associated with an exacerbated inflammatory response and with anemia and pancytopenia if bone marrow is affected. So far, the effect of a possible interaction of Histoplasma with stem cells present in the bone marrow is unknown.ObjectivesTo examine, in vitro, the immunomodulatory effects of MSCs in response to H. capsulatum infection.MethodsMSCs were obtained from bone marrow of C57BL/6 male mice; after isolation and purification, they were induced to mesodermal lineages and characterized by flow cytometry. Later, the basal expression of toll-like receptor (TLR)-2, TLR4, and Dectin-1 was determined using flow cytometry. MSCs were infected with H. capsulatum yeasts (isolate CIB 1980) in a multiplicity of infection (MOI) of 5 and incubated for 24 h. In addition, some of the co-cultures were previously treated with specific blocking antibodies for TLR2 and TLR4 or with a blocking peptide specific for Dectin-1 (CLEC7A). Furthermore, phagocytosis, microbicidal, and cell proliferation assays were done, and the expression of the genes encoding the cytokines IL-1β, IL-6, IL-10, IL-17, TNF-α, and TGF-β as well as of those for arginase-1 and iNOS were assessed.ResultsWe observed that H. capsulatum has the capability to adhere and internalize within these MSCs; nonetheless, this process did not affect the survival of the fungus. The interaction of H. capsulatum with MSCs induced a slight but significantly increased expression of TLR2 but not TLR4 nor Dectin-1. In addition, this fungal interaction significantly induced an augmented expression of IL-6 and a decrease in the expression of IL-1β, IL-17, TNF-α, TGF-β, as well as the immune mediators Arg-1 and iNOS. Interestingly, blockade of these receptors did not affect phagocytosis, but increased IL-1β, IL-17, and TNF-α expression and reduced the expression of IL-6. Noteworthy, H. capsulatum induced apoptosis and inhibited the proliferation of these stem cells; furthermore, this fungus significantly reduced the expression of genes related to adipogenic differentiation and increased the expression of genes related to the osteogenic differentiation process.ConclusionsThe above results indicate that MSCs do not exert a notable antifungal effect against H. capsulatum; on the contrary, this fungal pathogen not only modulates the expression of inflammatory mediators in MSCs, by a mechanism dependent on TLR2, TLR4, and Dectin-1, but also affects their viability and their ability to differentiate into a different type of specialized cells. These events could, in principle, affect both hematopoiesis and the immune response in the infected host, and in addition, these stem cells may provide a niche for this fungus, allowing it to persist and evade host immunity.

Investigation of PTC124-mediated translational readthrough in a retinal organoid model of AIPL1-associated Leber congenital amaurosis.

SummaryLeber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents.

Human Fetal Cartilage-Derived Progenitor Cells Exhibit Anti-Inflammatory Effect on IL-1β-Mediated Osteoarthritis Phenotypes In Vitro.

In this study, we have investigated whether human fetal cartilage progenitor cells (hFCPCs) have anti-inflammatory activity and can alleviate osteoarthritis (OA) phenotypes in vitro. hFCPCs were stimulated with various cytokines and their combinations and expression of paracrine factors was examined to find an optimal priming factor. Human chondrocytes or SW982 synoviocytes were treated with interleukin-1β (IL-1β) to produce OA phenotype, and co-cultured with polyinosinic-polycytidylic acid (poly(I-C))-primed hFCPCs to address their anti-inflammatory effect by measuring the expression of OA-related genes. The effect of poly(I-C) on the surface marker expression and differentiation of hFCPCs into 3 mesodermal lineages was also examined. Among the priming factors tested, poly(I-C) (1µg/mL) most significantly induced the expression of paracrine factors such as indoleamine 2,3-dioxygenase, histocompatibility antigen, class I, G, tumor necrosis factor- stimulated gene-6, leukemia inhibitory factor, transforming growth factor-β1 and hepatocyte growth factor from hFCPCs. In the OA model in vitro, co-treatment of poly(I-C)-primed hFCPCs significantly alleviated IL-1β-induced expression of inflammatory factors such as IL-6, monocyte chemoattractant protein-1 and IL-1β, and matrix metalloproteinases in SW982, while it increased the expression of cartilage extracellular matrix such as aggrecan and collagen type II in human chondrocytes. We also found that treatment of poly(I-C) did not cause significant changes in the surface marker profile of hFCPCs, while showed some changes in the 3 lineages differentiation. These results suggest that poly(I-C)-primed hFCPCs have an ability to modulate inflammatory response and OA phenotypes in vitro and encourage further studies to apply them in animal models of OA in the future.

Human mesenchymal amniotic fluid stem cells reveal an unexpected neuronal potential differentiating into functional spinal motor neurons.

Human amniotic fluids stem cells (hAFSCs) can be easily isolated from the amniotic fluid during routinely scheduled amniocentesis. Unlike hiPSCs or hESC, they are neither tumorigenic nor immunogenic and their use does not rise ethical or safety issues: for these reasons they may represent a good candidate for the regenerative medicine. hAFSCs are generally considered multipotent and committed towards the mesodermal lineages; however, they express many pluripotent markers and share some epigenetic features with hiPSCs. Hence, we hypothesized that hAFSCs may overcome their mesodermal commitment differentiating into to ectodermal lineages. Here we demonstrated that by the sequential exposure to specific factors, hAFSCs can give rise to spinal motor neurons (MNs), as evidenced by the gradual gene and protein upregulation of early and late MN markers (PAX6, ISL1, HB9, NF-L, vAChT). When co-cultured with myotubes, hAFSCs-derived MNs were able to create functional neuromuscular junctions that induced robust skeletal muscle contractions. These data demonstrated the hAFSCs are not restricted to mesodermal commitment and can generate functional MNs thus outlining an ethically acceptable strategy for the study and treatment of the neurodegenerative diseases.

Placenta‐Derived Mesenchymal Stem Cells for Treatment of Diseases: A Clinically Relevant Source

AbstractThe placenta is an organ that is discarded after childbirth. Recently, mesenchymal stem cells (MSCs) isolated from various parts of the placenta (PMSCs) are established as a rich, allogeneic, and sustainable source of MSCs in comparison to bone marrow MSCs (BM‐MSCs). PMSCs can be banked postnatally for future autologous and allogeneic applications in the treatment of diseases. PMSCs can be categorized as an intermediary between BM‐MSCs and embryonic stem cells (ESCs) as it is devoid of adverse aspects associated with the employment of ESCs accompanied with primitive and enhanced properties in comparison to BM‐MSCs. PMSCs are employed in the treatment of various diseases including cancer, neurological, bone, and cardiovascular disorders. This utility of PMSCs is due to its superior inherent characteristics in comparison to BM‐MSCs, which renders PMSCs more attractive for clinical translation. Herein, this review describes the superior inherent characteristics of PMSCS in contrast to BM‐MSCs including accessibility, higher expansion abilities, enhanced immunomodulatory and immunosuppressive properties, ability to differentiate beyond mesodermal lineages, pathotropic and regenerative abilities, lower immunogenicity, and anti‐cancer strategy that are correlated to its therapeutic application in the treatment of various diseases including corona virus infection started in 2019 in recent preclinical and preclinical studies.

Deducing Insulin-Producing Cells from Goat Adipose Tissue-Derived Mesenchymal Stem Cells.

Mesenchymal stem cell is a potent tool for regenerative medicine against control of incurable diseases in human and animals. Diabetes mellitus is one such condition marked with the blood glucose is high due to lack of insulin (INS) hormone secreted by the pancreatic cells. Rare, but sporadic, cases of dysfunctional pancreatic cells in goat as well as the promises of stem cell therapy as an off-the-shelf medicine prompted us to explore the potential of adipose-derived goat mesenchymal stem cells (AD-MSCs) to transdifferentiate into pancreatic islet-like cells. We isolated, in vitro cultured, and characterized the AD-MSCs by expression of MSC-specific markers and differentiation into multiple mesodermal lineage cells. The characterized AD-MSCs were in vitro transdifferentiated into INS-producing islet-like cells using a cocktail of glucose, nicotinamide, activin-A, exendin-4, pentagastrin, retinoic acid, and mercaptoethanol in 3 weeks. The transdifferentiated islet-like cells demonstrated the expression of pancreatic endoderm-specific transcripts PDX1, NGN3, PAX6, PAX4, ISL1, and GLUT2 as well as protein expression of pancreatic and duodenal homeobox 1 (PDX1), INS, and Islets 1 (ISL1). The islet-like cells also demonstrated the significant glucose-dependent INS release with respect to the course of transdifferentiation regime. The study envisaged to create the building material for basic research into mechanism of glucose homeostasis, which may pave road for developments in diabetes drug discovery and regenerative therapies.

Direct reprogramming of adult adipose-derived regenerative cells toward cardiomyocytes using six transcriptional factors

SummaryIt is widely accepted that adipose-derived regenerative cells (ADRCs) can differentiate into mesodermal lineage cells. However, reprogramming adult ADRCs into mature cardiomyocytes is challenging. We investigated the induction of myocardial differentiation in ADRCs via direct reprogramming using lentiviral gene transfer. First, we identified candidate transcriptional factors by performing RNA sequencing and ultimately confirmed that the combination of six unique factors (Baf60c, Gata4, Gata6, Klf15, Mef2a, and Myocd) could efficiently express enhanced green fluorescent protein (GFP) in ADRCs isolated from adult alpha-myosin heavy chain promoter-driven GFP transgenic mice. The GFP-positive ADRCs induced by six factors (6F-ADRCs) expressed multiple cardiac genes and revealed cardiac differentiation in bioinformatic analysis. Moreover, injection of 6F-ADRCs into acute myocardial infarcted tissues in vivo resulted in the improvement of survival rate, fractional shortening, and reduction of infarction scar area. This study provides an alternative method for direct reprogramming of adult ADRCs into cardiomyocytes.

Dissecting signaling hierarchies in the patterning of the mouse primitive streak using micropatterned EpiLC colonies

SummaryEmbryo studies have established that the patterning of the mouse gastrula depends on a regulatory network in which the WNT, BMP, and NODAL signaling pathways cooperate, but aspects of their respective contributions remain unclear. Studying their impact on the spatial organization and developmental trajectories of micropatterned epiblast-like cell (EpiLC) colonies, we show that NODAL is required prior to BMP action to establish the mesoderm and endoderm lineages. The presence of BMP then forces NODAL and WNT to support the formation of posterior primitive streak (PS) derivatives, while its absence allows them to promote that of anterior PS derivatives. Also, a Nodal mutation elicits more severe patterning defects in vitro than in the embryo, suggesting that ligands of extra-embryonic origin can rescue them. These results support the implication of a combinatorial process in PS patterning and illustrate how the study of micropatterned EpiLC colonies can complement that of embryos.