Mesodermal Lineages Research Articles

Changes in chromatin accessibility landscape and histone H3 core acetylation during valproic acid-induced differentiation of embryonic stem cells

Directed differentiation of mouse embryonic stem cells (mESCs) or induced pluripotent stem cells (iPSCs) provides powerful models to dissect the molecular mechanisms leading to the formation of specific cell lineages. Treatment with histone deacetylase inhibitors can significantly enhance the efficiency of directed differentiation. However, the mechanisms are not well understood. Here, we use CUT&RUN in combination with ATAC-seq to determine changes in both histone modifications and genome-wide chromatin accessibility following valproic acid (VPA) exposure. VPA induced a significant increase in global histone H3 acetylation (H3K56ac), a core histone modification affecting nucleosome stability, as well as enrichment at loci associated with cytoskeletal organization and cellular morphogenesis. In addition, VPA altered the levels of linker histone H1 subtypes and the total histone H1/nucleosome ratio indicative of initial differentiation events. Notably, ATAC-seq analysis revealed changes in chromatin accessibility of genes involved in regulation of CDK serine/threonine kinase activity and DNA duplex unwinding. Importantly, changes in chromatin accessibility were evident at several key genomic loci, such as the pluripotency factor Lefty, cardiac muscle troponin Tnnt2, and the homeodomain factor Hopx, which play critical roles in cardiomyocyte differentiation. Massive parallel transcription factor (TF) footprinting also indicates an increased occupancy of TFs involved in differentiation toward mesoderm and endoderm lineages and a loss of footprints of POU5F1/SOX2 pluripotency factors following VPA treatment. Our results provide the first genome-wide analysis of the chromatin landscape following VPA-induced differentiation in mESCs and provide new mechanistic insight into the intricate molecular processes that govern departure from pluripotency and early lineage commitment.

Characterization and differentiation potential of mesenchymal stem cells isolated from multiple canine adipose tissue sources

BackgroundMesenchymal stem cells (MSCs) are undifferentiated cells that can give rise to a mesoderm lineage. Adipose-derived MSCs are an easy and accessible source for MSCs isolation, although each source of MSC has its own advantages and disadvantages. Our study identifies a promising source for the isolation and differentiation of canines MSCs. For this purpose, adipose tissue from inguinal subcutaneous (SC), perirenal (PR), omental (OM), and infrapatellar fat pad (IPFP) was isolated and processed for MSCs isolation. In the third passage, MSCs proliferation/metabolism, surface markers expression, in vitro differentiation potential and quantitative reverse transcription PCR (CD73, CD90, CD105, PPARγ, FabP4, FAS, SP7, Osteopontin, and Osteocalcin) were evaluated.ResultsOur results showed that MSCs derived from IPFP have a higher proliferation rate, while OM-derived MSCs have higher cell metabolism. In addition, MSCs from all adipose tissue sources showed positive expression of CD73 (NT5E), CD90 (THY1), CD105 (ENDOGLIN), and very low expression of CD45. The isolated canine MSCs were successfully differentiated into adipogenic and osteogenic lineages. The oil-red-O quantification and adipogenic gene expression (FAS, FabP4, and PPARγ) were higher in OM-derived cells, followed by IPFP-MSCs. Similarly, in osteogenic differentiation, alkaline phosphatase activity and osteogenic gene (SP7 and Osteocalcin) expression were higher in OM-derived MSCs, while osteopontin expression was higher in PR-derived MSCs.ConclusionIn summary, among all four adipose tissue sources, OM-derived MSCs have better differentiation potential toward adipo- and osteogenic lineages, followed by IPFP-MSCs. Interestingly, among all adipose tissue sources, MSCs derived from IPFP have the maximum proliferation potential. The characterization and differentiation potential of canine MSCs isolated from four different adipose tissue sources are useful to assess their potential for application in regenerative medicine.

Computational and in vitro validation of cardiogenic induction of quercetin on adipose-derived mesenchymal stromal cells through the inhibition of Wnt and non-Smad-dependent TGF-β pathways.

Exploiting human mesenchymal stem cells (hMSCs) was proposed as a promising therapeutic approach for cardiovascular disease due to their capacity to differentiate into cardiac cells. Though modulation of the intracellular signaling pathways dominantly WNT/β catenin and transforming growth factor-β (TGF-β) have been reported to promote differentiation of hMSCs into cardiomyocytes in the prevailing literature, a safe and reproducible system for their clinical application has not yet turned into reality. In the present study, the molecular docking-based strategy was first applied for evaluating the potency of some natural phenolic compounds in the modulation of Wnt and TGF-β signaling pathways using a vital class of crystallographic protein structures of WNT signaling regulators such as Frizzled, Disheveled, GSK3-β, β-catenin, LRP 5/6 extracellular domain, Tankyrase and their variety of active pockets. Then, the impacts of plant-derived chemical compounds on the regulation of the relevant signals for the differentiation of hMSCs into the definitive mesoderm lineage and cardiac progenitors were assessed in vitro. Data obtained revealed the synergistic activity of Wnt and TGF-β superfamily to direct cardiac differentiation in human cardiogenesis by comparing cardiac gene expression in the presence and absence of the TGF-β inhibitors. We found that the inhibitory effect of canonical Wnt/β-catenin is sufficient to cause proper cardiomyocyte differentiation, but the TGF-β pathway plays a vital role in enhancing the expression of the cardiomyocyte-specific marker (cTnT). It was found that quercetin, a p38MAPK inhibitor with the high energy dock to the active pocket of Wnt receptors, promotes cardiac differentiation via the inhibition of both Wnt and non-Smad TGF-β pathways. Altogether, data presented here can contribute to the development of a feasible and efficient cardiac differentiation protocol as an "off-the-shelf" therapeutic source using novel natural agents for cardiac repair or regeneration.

Abstract 13303: Identification of Critical Six Transcriptional Factors for Reprogramming of Mouse Adipose-Derived Regenerative Cells Toward Cardiomyocytes and Its Therapeutic Effect

Background: Adipose-derived regenerative cells (ADRCs) are promising stem cells for regenerative medicine that are easy to harvest from adipose tissues and can expand in vitro . While ADRCs can differentiate into mesodermal lineage cells including adipocytes, osteocytes, and chondrocytes, little is known about differentiating adult ADRCs into mature cardiomyocytes. Objective: The aim of this study was to explore how to differentiate adult mouse ADRCs into cardiomyocytes efficiently and test their effect on the myocardial infarction mouse model in vivo . Method and Results: We performed differential expression analysis to identify transcriptional factors which were upregulated in developing mice heart (E11.5) compared with ADRCs. We systemically screened the combinations of the required transcription factors in ADRCs derived from αMHC-EGFP reporter mice. Here, we identified the following six genes; Baf60c , Gata4 , Gata6 , Klf15 , Mef2a , Myocd , not more or less, as the best combination to efficiently induce αMHC in ADRCs. Fluorescence-activated cell sorting analysis showed that 3.0±0.61% (n=3) ADRCs were positive for αMHC after introducing these six factors. In addition, quantitative PCR demonstrated that several other marker genes of mature cardiomyocytes, including Actc1 , Tnnt2 , Ttn , Fhod3 , were confirmed to be upregulated by six factors. Immunocytochemical staining showed that most of the induced ADRCs had fibrous conformation of sarcomeric α-actin. Moreover, the gene expression pattern of the cells showed upregulation of genes associated with cardiac muscle cell differentiation. Finally, injection of the induced ADRCs into acute myocardial infarction lesions in vivo resulted in the improvement of survival rate (p=0.028, n=32), fractional shortening (p=0.012, n=7, 15), and the reduction of infarction scar area compared to the injection of ADRCs without induction (p=0.029, n=7). The induced ADRCs could survive in situ and continued to express the cardiac troponin T protein for four weeks after injection. Conclusions: The combination of six transcriptional factors could induce cardiomyocyte-like gene expression patterns on adult ADRCs. The induced ADRCs could have a therapeutic effect on an experimental ischemic heart model in vivo .

The Clinical Trials of Mesenchymal Stromal Cells Therapy.

Mesenchymal stromal cells (MSCs) are a heterogeneous population of adult stem cells, which are multipotent and possess the ability to differentiate/transdifferentiate into mesodermal and nonmesodermal cell lineages. MSCs display broad immunomodulatory properties since they are capable of secreting growth factors and chemotactic cytokines. Safety, accessibility, and isolation from patients without ethical concern make MSCs valuable sources for cell therapy approaches in autoimmune, inflammatory, and degenerative diseases. Many studies have been conducted on the application of MSCs as a new therapy, but it seems that a low percentage of them is related to clinical trials, especially completed clinical trials. Considering the importance of clinical trials to develop this type of therapy as a new treatment, the current paper is aimed at describing characteristics of MSCs and reviewing relevant clinical studies registered on the NIH database during 2016-2020 to discuss recent advances on MSC-based therapeutic approaches being used in different diseases.

A small molecule modulating monounsaturated fatty acids and Wnt signaling confers maintenance to induced pluripotent stem cells against endodermal differentiation

BackgroundStearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. Through the fatty acid acylation process, this enzyme orchestrates post-translational modifications to proteins involved in cell development and differentiation. In this study, we used biochemical methods, immunostaining, and covalent labeling to evaluate whether a small molecule modulating unsaturated fatty acids can influence the early endodermal differentiation of human-induced pluripotent stem cells (iPSCs).MethodsThe hiPSCs were cultured in an endoderm-inducing medium containing activin A and defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The cell cycles and the yields of the three germ layers (endoderm, mesoderm, and ectoderm) were assessed using flow cytometry. The expression of endoderm and pluripotency markers and the expressions of Wnt signaling pathway proteins were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction.ResultsWhen SCD1 was inhibited on the first day, the population of cells with endodermal features decreased at the end of differentiation. Moreover, early SCD1 inhibition preserved the properties of hiPSCs, preventing their shift toward mesodermal or ectodermal lineage. Also, first-day-only treatment of cells with the SCD1 inhibitor decreased β-catenin gene expression and the intensity of fluorescent emission in the click chemistry assay. The cells were effectively rescued from these effects by cotreatment with oleate. Late treatment with the inhibitor in the two subsequent days of endoderm induction did not have any significant effects on endoderm-specific markers or fluorescent intensity. Reproducible results were also obtained with human embryonic stem cells.ConclusionThe small molecule SCD1 inhibitor attenuates the Wnt/β-catenin signaling pathway, conferring the maintenance of hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in the endoderm commitment of pluripotent stem cells may be of importance in disorders of endoderm-derived organs and dysregulated metabolism.The schematic representation of the study design and main results. Activin A induces endoderm features through Smad2/3/4 and increases the expression of SCD1. SCD1 can produce MUFAs and subsequently modify the Wnt molecules. MUFA acylated/activated Wnts are secreted to interact with corresponding receptors on the target cells. β-catenin accumulates in the cytoplasm and is translocated into the nucleus after the interaction of Wnt with the receptor. Then, β-catenin increases the expression of the endoderm markers Sox17 and CXCR4.

Distinct Mitochondrial Remodeling During Mesoderm Differentiation in a Human-Based Stem Cell Model.

Given the considerable interest in using stem cells for modeling and treating disease, it is essential to understand what regulates self-renewal and differentiation. Remodeling of mitochondria and metabolism, with the shift from glycolysis to oxidative phosphorylation (OXPHOS), plays a fundamental role in maintaining pluripotency and stem cell fate. It has been suggested that the metabolic “switch” from glycolysis to OXPHOS is germ layer-specific as glycolysis remains active during early ectoderm commitment but is downregulated during the transition to mesoderm and endoderm lineages. How mitochondria adapt during these metabolic changes and whether mitochondria remodeling is tissue specific remain unclear. Here, we address the question of mitochondrial adaptation by examining the differentiation of human pluripotent stem cells to cardiac progenitors and further to differentiated mesodermal derivatives, including functional cardiomyocytes. In contrast to recent findings in neuronal differentiation, we found that mitochondrial content decreases continuously during mesoderm differentiation, despite increased mitochondrial activity and higher levels of ATP-linked respiration. Thus, our work highlights similarities in mitochondrial remodeling during the transition from pluripotent to multipotent state in ectodermal and mesodermal lineages, while at the same time demonstrating cell-lineage-specific adaptations upon further differentiation. Our results improve the understanding of how mitochondrial remodeling and the metabolism interact during mesoderm differentiation and show that it is erroneous to assume that increased OXPHOS activity during differentiation requires a simultaneous expansion of mitochondrial content.

Human pluripotent stem cell-derived cartilaginous organoids promote scaffold-free healing of critical size long bone defects

BackgroundBones have a remarkable capacity to heal upon fracture. Yet, in large defects or compromised conditions healing processes become impaired, resulting in delayed or non-union. Current therapeutic approaches often utilize autologous or allogeneic bone grafts for bone augmentation. However, limited availability of these tissues and lack of predictive biological response result in limitations for clinical demands. Tissue engineering using viable cell-based implants is a strategic approach to address these unmet medical needs.MethodsHerein, the in vitro and in vivo cartilage and bone tissue formation potencies of human pluripotent stem cells were investigated. The induced pluripotent stem cells were specified towards the mesodermal lineage and differentiated towards chondrocytes, which subsequently self-assembled into cartilaginous organoids. The tissue formation capacity of these organoids was then challenged in an ectopic and orthotopic bone formation model.ResultsThe derived chondrocytes expressed similar levels of collagen type II as primary human articular chondrocytes and produced stable cartilage when implanted ectopically in vivo. Upon targeted promotion towards hypertrophy and priming with a proinflammatory mediator, the organoids mediated successful bridging of critical size long bone defects in immunocompromised mice.ConclusionsThese results highlight the promise of induced pluripotent stem cell technology for the creation of functional cartilage tissue intermediates that can be explored for novel bone healing strategies.

Characterization of the Nonendocrine Cell Populations in Human Embryonic Stem Cell-Derived (hESC) Islet-Like Clusters Posttransplantation.

Islet-like clusters derived from human embryonic stem cells (hESC) hold the potential to cure type 1 diabetes mellitus. Differentiation protocols of islet-like clusters lead to the generation of minor fractions of nonendocrine cells, which are mainly from endodermal and mesodermal lineages, and the risk of implanting these is unclear. In the present study, the histogenesis and the tumorigenicity of nonendocrine cells were investigated in vivo. Immunodeficient mice were implanted under the kidney capsule with islet-like clusters which were derived from differentiation of cells batches with either an intermediate or poor cell purity and followed for 8 or 26 weeks. Using immunohistochemistry and other techniques, it was found that the intermediate differentiated cell implants had limited numbers of small duct-like cysts and nonpancreatic tissue resembling gastrointestinal and retinal pigmented epithelium. In contrast, highly proliferative cystic teratomas were found at a high incidence at the implant site after 8 weeks, only in the animals implanted with the poorly differentiated cells. These findings indicate that the risk for teratoma formation and the amount of nonpancreatic tissue can be minimized by careful in-process characterization of the cells and thus highlights the importance of high purity at transplantation and a thorough ex-vivo characterization during cell product development.

Therapeutic Implications of Mesenchymal Stromal Cells and Their Extracellular Vesicles in Autoimmune Diseases: From Biology to Clinical Applications.

Mesenchymal stromal cells (MSCs) are perivascular multipotent stem cells originally identified in the bone marrow (BM) stroma and subsequently in virtually all vascularized tissues. Because of their ability to differentiate into various mesodermal lineages, their trophic properties, homing capacity, and immunomodulatory functions, MSCs have emerged as attractive candidates in tissue repair and treatment of autoimmune disorders. Accumulating evidence suggests that the beneficial effects of MSCs may be primarily mediated via a number of paracrine-acting soluble factors and extracellular vesicles (EVs). EVs are membrane-coated vesicles that are increasingly being acknowledged as playing a key role in intercellular communication via their capacity to carry and deliver their cargo, consisting of proteins, nucleic acids, and lipids to recipient cells. MSC-EVs recapitulate the functions of the cells they originate, including immunoregulatory effects but do not seem to be associated with the limitations and concerns of cell-based therapies, thereby emerging as an appealing alternative therapeutic option in immune-mediated disorders. In the present review, the biology of MSCs will be outlined and an overview of their immunomodulatory functions will be provided. In addition, current knowledge on the features of MSC-EVs and their immunoregulatory potential will be summarized. Finally, therapeutic applications of MSCs and MSC-EVs in autoimmune disorders will be discussed.

From Bipotent Neuromesodermal Progenitors to Neural-Mesodermal Interactions during Embryonic Development.

To ensure the formation of a properly patterned embryo, multiple processes must operate harmoniously at sequential phases of development. This is implemented by mutual interactions between cells and tissues that together regulate the segregation and specification of cells, their growth and morphogenesis. The formation of the spinal cord and paraxial mesoderm derivatives exquisitely illustrate these processes. Following early gastrulation, while the vertebrate body elongates, a population of bipotent neuromesodermal progenitors resident in the posterior region of the embryo generate both neural and mesodermal lineages. At later stages, the somitic mesoderm regulates aspects of neural patterning and differentiation of both central and peripheral neural progenitors. Reciprocally, neural precursors influence the paraxial mesoderm to regulate somite-derived myogenesis and additional processes by distinct mechanisms. Central to this crosstalk is the activity of the axial notochord, which, via sonic hedgehog signaling, plays pivotal roles in neural, skeletal muscle and cartilage ontogeny. Here, we discuss the cellular and molecular basis underlying this complex developmental plan, with a focus on the logic of sonic hedgehog activities in the coordination of the neural-mesodermal axis.

Comparative analysis of human induced pluripotent stem cell-derived mesenchymal stem cells and umbilical cord mesenchymal stem cells.

Generation of induced pluripotent stem cells (iPSCs) and their differentiation into mesenchymal stem/stromal cells (iMSCs) have created exciting source of cells for autologous therapy. In this study, we have compared the therapeutic potential of iMSCs generated from urinary epithelial (UE) cells with the available umbilical cord MSCs (UC‐MSCs). For this, adult UE cells were treated with the mRNA of pluripotent genes (OCT4, NANOG, SOX2, KLF4, MYC and LIN28) and a cocktail of miRNAs under specific culture conditions for generating iPSCs. Our non‐viral and mRNA‐based treatment regimen demonstrated a high reprogramming efficiency to about 30% at passage 0. These UE‐iPSCs were successfully differentiated further into ectoderm, endoderm and mesoderm lineage of cells. Moreover, these UE‐iPSCs were subsequently differentiated into iMSCs and were compared with the UC‐MSCs. These iMSCs were capable of differentiating into osteocytes, chondrocytes and adipocytes. Our qRT‐PCR and Western blot data showed that the CD73, CD90 and CD105 gene transcripts and proteins were highly expressed in iMSCs and UC‐MSCs but not in other cells. The comparative qRT‐PCR data showed that the iMSCs maintained their MSC characteristics without any chromosomal abnormalities even at later passages (P15), during which the UC‐MSCs started losing their MSC characteristics. Importantly, the wound‐healing property demonstrated through migration assay was superior in iMSCs when compared to the UC‐MSCs. In this study, we have demonstrated an excellent non‐invasive and pain‐free method of obtaining iMSCs for regenerative therapy. These homogeneous autologous highly proliferative iMSCs may provide an alternative source of cells to UC‐MSCs for treating various diseases.

Dynamics of primitive streak regression controls the fate of neuromesodermal progenitors in the chicken embryo.

In classical descriptions of vertebrate development, the segregation of the three embryonic germ layers completes by the end of gastrulation. Body formation then proceeds in a head to tail fashion by progressive deposition of lineage-committed progenitors during regression of the primitive streak (PS) and tail bud (TB). The identification by retrospective clonal analysis of a population of neuromesodermal progenitors (NMPs) contributing to both musculoskeletal precursors (paraxial mesoderm) and spinal cord during axis formation challenged these notions. However, classical fate mapping studies of the PS region in amniotes have so far failed to provide direct evidence for such bipotential cells at the single-cell level. Here, using lineage tracing and single-cell RNA sequencing in the chicken embryo, we identify a resident cell population of the anterior PS epiblast, which contributes to neural and mesodermal lineages in trunk and tail. These cells initially behave as monopotent progenitors as classically described and only acquire a bipotential fate later, in more posterior regions. We show that NMPs exhibit a conserved transcriptomic signature during axis elongation but lose their epithelial characteristicsin the TB. Posterior to anterior gradients of convergence speed and ingression along the PS lead to asymmetric exhaustion of PS mesodermal precursor territories. Through limited ingression and increased proliferation, NMPs are maintained and amplified as a cell population which constitute the main progenitors in the TB. Together, our studies provide a novel understanding of the PS and TB contribution through the NMPs to the formation of the body of amniote embryos.

Comparison of characteristics of mesenchymal stem cells from bovine

Mesenchymal stem cells (MSCs) are multipotent cells capable of replicating as undifferentiated cells, and thus hold therapeutic implications in field of regenerative medicine and reproductive biotechnology. In the present study, we compared the stem cell properties of bovine ear skin tissue (ESK)- and nasal mucosa (NM)-derived MSCs. Bovine ESK-MSCs and NM-MSCs were successfully isolated by collagenase digestion and maintained proliferative capacity during the 20 consecutive passages. Both ESK-MSCs and NM-MSCs showed similar morphology and expressed common cell surface markers (CD29, CD44, CD90, and CD105). Also, we compared differentiation potentials of bovine ESK-MSCs and NM-MSCs into osteogenic, adipogenic, and chondrogenic lineages through specific staining and quantitative real-time RT-PCR. As results, bovine ESK-MSCs and NM-MSCs could differentiate into mesodermal cell lineages. However, bovine ESK-MSCs and NM-MSCs exhibited difference in expression of differentiation-related specific markers. Specifically, NM-MSCs exhibited increased expression levels of osteocalcin, peroxisome proliferator-activated receptor gamma, and aggrecan compared to ESK-MSCs. Also, ESK-MSCs exhibited increased expression levels of collagen type I, II, and lipoprotein lipase compared to NM-MSCs. We suggest that the nasal mucosa of bovine could be used as a source of bovine MSCs.

Defining the Role of a GATA-2 Enhancer during Hematopoietic Differentiation of Human iPSCs

Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, USARationale : Differentiation of hematopoietic stem cells from human induced pluripotent stem cells (iPSCs) has the potential to provide novel therapeutic options for a wide range of blood diseases; however, despite significant progress that has been made in driving iPSC differentiation, issues with limited differentiation potential and engraftment capability in vivo still persist. A better understanding of the key regulators of hematopoiesis is essential for the development of improved differentiation protocols. GATA-2, a transcription factor shown to be essential for hematopoiesis, has been examined to determine its role in both hematopoiesis and endothelial cell development. Mouse studies have described an early developmental Gata-2 hematopoietic-specific enhancer located 5′ to its distal first exon. Based on homology within this regulatory domain between mouse and human sequences, we hypothesize that this cis-regulatory region plays an essential role in human hematopoietic expression of GATA-2 and in endothelial-to-hematopoietic transition.Methods : In this study, we use a genome editing approach and an in vitro human iPSC differentiation system to examine our hypothesis. A pair of CRISPR/Cas9 guide RNA was designed to recognize sequences of a DNase I hypersensitive site (DHS) within the putative enhancer region immediately upstream of the GATA-2 distal first exon. Human iPSCs were treated with CRISPR/Cas9 to create cell lines that have a deletion at this DHS. These genetically modified iPSC lines were compared to their isogenic parental controls, to examine functional consequences of the deletion. Both adherent culture-based and spin-embryoid body formation-based protocols were used for mesodermal differentiation, endothelial specification and hematopoietic induction.Results : Screening of human iPSC clones after co-transfection of Cas9 and guide RNA-expression vectors has identified clones with ~400 bp deletion of the DNase I hypersensitive site that is conserved among vertebrates. When maintained in iPSC expansion condition, these iPSC clones display typical undifferentiated human iPSC morphology and expression of pluripotency-related markers such as TRA-1-60. Upon treatment with a Wnt signaling activator, BMP4, bFGF and VEGF, the genetically modified cells showed similar differentiation efficiency, compared to parental controls, to mesoderm and endothelial lineages. However, they exhibit a lower proliferation rate in endothelial expansion conditions. Preliminary studies also showed a deficiency of the modified cells in their ability to form capillary-like structures in tube formation assays. In hematopoietic differentiation experiments using the spin-EB method, these cells showed very low viability upon hematopoietic induction and failed to produce hematopoietic progenitor cells. These results suggest that the DNase I hypersensitive site upstream of GATA2 distal first exon plays an important role in human endothelial cell function including transition from hemogenic endothelium to hematopoietic cells upon stimulation.Currently, experiments are being conducted to characterize the functionality of endothelial-like cells and to confirm previous observations in additional iPSC lines. Differential expression of GATA-2 between parental and genome edited cells at various developmental stages is being determined. Hematopoietic differentiation capacity of these iPSCs by stromal cell co-culture methods is being used to confirm observations from the spin-EB protocol. Defining the functional outcome of this targeted deletion, across several iPSC lines, will provide a system for understanding molecular regulations in the first steps of human hematopoietic stem cell generation. DisclosuresNo relevant conflicts of interest to declare.

Concurrent Ex Vivo Differentiation of a Vascular Niche and Hematopoietic Stem/Progenitor Cells from Human iPSCs

One of the most promising objectives of clinical hematology is to derive transplantable autologous hematopoietic stem/progenitor cells (HSPCs) ex vivo. Recent reports have described the generation of engraftable HSPCs via forced expression of key transcription factors in either endothelial cells, or hemogenic endothelium (HE) derived from induced pluripotent stem cells (iPSCs). While protocols for the generation of transgene-free engraftable HSPCs suitable for therapy remain unavailable, these studies, along with observations in the developing embryo, have shown the close association of HE populations with arterial vascular endothelium is of critical importance to HSPC production and maturation. To facilitate the development of a vascular niche supporting the emergence of transgene-free, functional HSPCs from human iPSCs, we developed a simple, monolayer-based, chemically-defined, and scalable differentiation protocol requiring no replating or embryoid body (EB) formation (commercially available as STEMdiffTM Hematopoietic Kit, Stem Cell Technologies). During the first 3 days, a mesodermal lineage specification was induced with morphogens (BMP4, bFGF and VEGF). For the subsequent 18 days, cells were further differentiated into HSPCs with the addition of hematopoietic cytokines (SCF, Flt3-L and TPO). Starting at day 5, an adherent monolayer composed in part of CD34+CD43- endothelial cells rapidly formed, followed by the initiation of endothelial-to-hematopoietic transition (EHT), as demonstrated by the emergence of round cells directly budding off the endothelial monolayer in time-lapse imaging studies (Panel A). Importantly, these cells were released within the culture supernatant and could be easily harvested by simple pipetting. Flow cytometric analysis of the supernatant cells indicated that the emerging cells initially had a predominant CD45-CD235a+ erythroid phenotype (maximum 69.9±12.4% at day 7), consistent with a primitive yolk-sac wave of hematopoiesis (PanelB). From day 7, CD45+CD34+ HSPCs increased gradually, with a maximum number (average 1x105 cells/cm2, n=10) observed at day 12, and a peak percentage at day 12 (47.4±12.4%) and 14 (48.8±9.9%) (Panel B). These cells could form colonies in clonogenic progenitor assays, albeit at reduced capacity compared to bona fide CD45+CD34+ cells. Strikingly, cells with a phenotype that enables the highest reported purity of human HSCs (CD34+ CD38- CD45RA- CD90+ CD49f+ Rholo) were highly enriched in the culture supernatant at day 10 through 14 (Panel C). However, these cells did not result in efficient, long-term engraftment in immuno-deficient (NSG) mice. To identify possible causes for the lack of durable repopulating potential of iPSC-derived HSPCs in this system, we further characterized the endothelial monolayer between day 5 and 12 of differentiation. We focused on recently described endothelial subsets within the CD34hiCD43- population, including the in vitro equivalent of definitive HE (CD34hi CD43- CD73- CD184- DLL4-), arterial vascular endothelium (CD34hi CD43- CD73mid CD184+), and venous vascular endothelium (CD34hi CD43- CD73hi CD184-) (Ditadi et al., Nat. Cell Biol 2015). At each timepoint analyzed, definitive HE comprised <1% of the total cell population (Panel D). Arterial vascular endothelium was also scarce, rising to a maximum of 6.6% at day 7 before declining to <1% by day 9 of culture (Panel D). Venous vascular endothelium accounted for approximately 2% of total cells (Panel D). The remaining cells of the monolayer expressed varying levels of CD34 and CD43, consistent with the recently demonstrated heterogeneity of cells undergoing EHT (Guibentif et al., Cell Rep 2017). Thus, our data suggest that the absence of a definitive HE population within a developing arterial niche, as occurs in the dorsal aorta (DA) during development, may explain in part the lack of engraftment potential of iPSC-derived HSPCs. The unique adaptable and scalable nature of this monolayer-based differentiation protocol will facilitate precise temporal activation or downregulation of signaling pathways essential for the generation of engraftable human HSPCs. Quantitative and temporal modulations of Wnt/Nodal pathways for mesodermal specification as well as VEGF/Notch pathways for arterial vascular endothelium are underway. [Display omitted] DisclosuresChen:Stem Cell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit. Boehm:StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit. Larochelle:Novartis: Research Funding; StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit; Novartis: Research Funding.

Unveiling Complexity and Multipotentiality of Early Heart Fields.

[Figure: see text].

Mesenchymal Stem Cells: A Novel Therapeutic Approach to Enhance Protective Immunomodulation and Erythropoietic Recovery in Malaria.

Mesenchymal stem cells (MSCs) are self-renewing, multi-potent heterogeneous stem cells that display strong tissue protective and restorative properties by differentiating into cells of the mesodermal lineages. In addition to multi-lineage differentiation capacity, MSCs play important roles in regulating immune responses, inflammation, and tissue regeneration. MSCs play a role in the outcome of the pathogenesis of several infectious diseases. A unique subset of MSCs accumulates in secondary lymphoid organs during malaria disease progression. These MSCs counteract the capacity of malaria parasites to subvert activating co-stimulatory molecules and to regulate expression of negative co-stimulatory molecules on T lymphocytes. Consequently, MSCs have the capacity to restore the functions of CD34+ haematopoietic cells and CD4+ and CD8+ T cells during malaria infection. These observations suggest that cell-based therapeutics for intervention in malaria may be useful in achieving sterile clearance and preventing disease reactivation. In addition, MSCs provide host protection against malaria by reprogramming erythropoiesis through accelerated formation of colony-forming-units-erythroid (CFU-E) cells in the bone marrow. These findings suggest that MSCs are positive regulators of erythropoiesis, making them attractive targets for treatment of malarial anemia. MSC-based therapies, unlike anti-malarial drugs, display therapeutic effects by targeting a large variety of cellular processes rather than a single pathway. In the present review we focus on these recent research findings and discuss clinical applications of MSC-based therapies for malaria.Graphical

Plant callus-derived shikimic acid regenerates human skin through converting human dermal fibroblasts into multipotent skin-derived precursor cells

BackgroundThe human skin-derived precursors (SKPs) are a good cell source for regeneration. However, the isolation of SKP from human skin is limited. To overcome this drawback, we hypothesized that the component of plant stem cells could convert human fibroblasts to SKPs.MethodsHuman dermal fibroblasts were treated with shikimic acid, a major component of Sequoiadendron giganteum callus extract. The characteristics of these reprogrammed cells were analyzed by qPCR, western blot, colony-forming assay, and immunofluorescence staining. Artificial human skin was used for CO2 laser-induced wound experiments. Human tissues were analyzed by immunohistochemistry.ResultsThe reprogrammed cells expressed nestin (a neural precursor-specific protein), fibronectin, and vimentin and could differentiate into the ectodermal and mesodermal lineage. Nestin expression was induced by shikimic acid through the mannose receptor and subsequent MYD88 activation, leading to P38 phosphorylation and then CREB binding to the nestin gene promoter. Finally, we confirmed that shikimic acid facilitated the healing of cut injury and enhanced dermal reconstruction in a human artificial skin model. Moreover, in a clinical study with healthy volunteers, plant callus extracts increased the expression of stem cell markers in the basal layer of the epidermis and collagen deposit in the dermis.ConclusionsThese results indicate that shikimic acid is an effective agent for tissue regeneration.

Comparative evaluation of isogenic mesodermal and ectomesodermal chondrocytes from human iPSCs for cartilage regeneration.

Generating phenotypic chondrocytes from pluripotent stem cells is of great interest in the field of cartilage regeneration. In this study, we differentiated human induced pluripotent stem cells into the mesodermal and ectomesodermal lineages to prepare isogenic mesodermal cell-derived chondrocytes (MC-Chs) and neural crest cell-derived chondrocytes (NCC-Chs), respectively, for comparative evaluation. Our results showed that both MC-Chs and NCC-Chs expressed hyaline cartilage-associated markers and were capable of generating hyaline cartilage-like tissue ectopically and at joint defects. Moreover, NCC-Chs revealed closer morphological and transcriptional similarities to native articular chondrocytes than MC-Chs. NCC-Ch implants induced by our growth factor mixture demonstrated increased matrix production and stiffness compared to MC-Ch implants. Our findings address how chondrocytes derived from pluripotent stem cells through mesodermal and ectomesodermal differentiation are different in activities and functions, providing the crucial information that helps make appropriate cell choices for effective regeneration of articular cartilage.