Expression Of Lineage-specific Markers Research Articles

Gain of 1q confers an MDM4-driven growth advantage to undifferentiated and differentiating hESC while altering their differentiation capacity

Gain of 1q is a highly recurrent chromosomal abnormality in human pluripotent stem cells. In this work, we show that gains of 1q impact the differentiation capacity to derivates of the three germ layers, leading to mis-specification to cranial placode and non-neural ectoderm during neuroectoderm differentiation. Also, we found a weaker expression of lineage-specific markers in hepatoblasts and cardiac progenitors. Competition assays show that the cells retain their selective advantage during differentiation, which is mediated by a higher expression of MDM4, a gene located in the common region of gain. MDM4 drives the winner phenotype of the mutant cells in both the undifferentiated and differentiating state by reducing the cells’ sensitivity to DNA damage through decreased p53-mediated apoptosis. Finally, we found that cell density in culture plays a key role in promoting the competitive advantage of the cells by increasing DNA damage.

Spontaneous Efficient Differentiation of Human Pluripotent Stem Cells (hPSC) Upon Co-culture of hPSCs with Human Neonatal Foreskin Fibroblasts in 3D.

Pluripotent stem cells (PSCs) form well-formed embryoid bodies (EBs) in 3D culture. These EBs are formed in culture media lacking leukemia inhibitory factor (LIF) or basic fibroblast growth factor (bFGF) in mouse and human PSCs, respectively. EBs are excellent technical tools for understanding developmental biology and inducing controlled differentiation in succeeding experimental steps. Technically speaking, EBs are spontaneously differentiated PSCs in 3D and exhibit all three lineages in a time-point/sequential manner. For example, ectoderm will form first, followed by mesoderm and endoderm. We have attempted to co-culture human neonatal foreskin-derived fibroblast cells in our laboratory with the PSCs first in 2D conditions followed by the induction of EBs (PSC+fibroblasts co-cultured) in low attachment dishes. We also performed spontaneous differentiation of such EBs (co-cultured with fibroblasts). We checked the presence of markers of various lineages, namely, ectoderm, mesoderm, and endoderm in days 6, 10, and 12day EBs. We have also compared the fibroblast co-cultured EBs, along with control EBs (derived from only PSCs). This co-culture system mimics the natural conditions of uterine implantation and the role of the endometrial fibroblasts in the induction of further embryonic development. The fibroblast co-cultured iPSC EBs had better roundness scores than the normal iPSC EBs and had a higher expression of lineage-specific markers.

O-072 The canonical Hippo signalling pathway controls the first lineage segregation in the human preimplantation embryo

Abstract Study question What is the role of the canonical Hippo signalling pathway during the first lineage segregation in the human preimplantation embryo? Summary answer Canonical Hippo signalling regulates the first lineage segregation by controlling the expression of trophectoderm (TE) and inner cell mass (ICM) associated genes. What is known already The Hippo signalling pathway is a conserved regulatory network that plays a critical role in the first lineage segregation in mouse, rat, bovine and human preimplantation embryos. This pathway consists of two distinct branches (canonical, non-canonical). Despite their shared role in lineage segregation, the respective functions of canonical and non-canonical Hippo signalling branches remain largely unexplored. Since these branches are regulated by distinct upstream mechanisms, they may have different roles in embryonic growth, function, and survival. Unravelling the distinct roles of these branches is essential for understanding the intricate mechanisms that govern early human embryonic development. Study design, size, duration This experimental study involved the functional characterization of the canonical Hippo signalling pathway by pharmacologically inhibiting the core kinase MST1/2. Eight-cell stage human embryos donated for research were warmed and incubated with or without the inhibitor, till completion of the first lineage segregation days post fertilization (dpf) 5. The control (n = 20) and treated embryos (n = 17) were evaluated based on embryonic development (kinetics, morphology), and expression of lineage-specific markers. Participants/materials, setting, methods The optimal concentration (1μΜ) of the highly selective XMU-MP-1 inhibitor targeting the MST1/2 core kinase of the canonical Hippo signalling pathway was established in titration experiments. Time-lapse imaging (Miri TL, Esco Medical) captured effects on embryonic development (kinetics, morphology). Changes in the main effectors of the Hippo signalling pathway (YAP1, p-YAP1 and WWTR1) and the expression of lineage-specific markers (ICM: NANOG, SOX2; TE: GATA3) were assessed through immunofluorescence imaging and confocal microscopy (LSM800, Zeiss). Main results and the role of chance For the first time, we provide a complete spatiotemporal analysis of the main effectors of the Hippo signalling pathway YAP1 and WWTR1. They are co-localised in the nuclei of outer cells in compacting (dpf3) and compacted (dpf4) embryos. In the dpf5 blastocyst, YAP1 and WWTR1 are detected in the nuclei of the TE cells. Phosphorylated effector p-YAP1, indicative of active Hippo signalling, was first detected in the inner cells of the compacted embryo (dpf4) and remained in the ICM of the dpf5 blastocyst, supporting the role of the Hippo signalling pathway in the first lineage segregation. Treatment of human embryos with XMU-MP-1 reduced p-YAP1 levels, confirming its specificity for MST1/2. Inhibition of the MST1/2 kinase had a significant effect on the developmental kinetics as treated embryos had lower blastocyst formation rates. Embryo morphology was significantly compromised as treated embryos formed ICMs with less cells compared to control embryos. Immunofluorescence analysis revealed that the XMU-MP-1 treatment led to the upregulation of YAP1 and upregulation of TE marker GATA3, while the ICM markers SOX2 and NANOG were downregulated. Overall, our data suggest that active Hippo signaling promotes ICM formation, while TE formation requires inactive Hippo signaling. Limitations, reasons for caution Caution is warranted due to potential non-specific effects of the XMU-MP-1 inhibitor, although this compound has been shown in other models to be very specific. Further studies should explore off-target impacts to ensure result reliability. Wider implications of the findings Understanding the signalling pathways that regulate human preimplantation embryo development is fundamental for understanding embryonic growth, function, and survival, and can also help improve IVF success rates by addressing deficiencies that lead to embryo developmental arrest. Trial registration number Not applicable

Small Molecules Promote the Rapid Generation of Dental Epithelial Cells from Human-Induced Pluripotent Stem Cells.

Human-induced pluripotent stem cells (hiPSCs) offer a promising source for generating dental epithelial (DE) cells. Whereas the existing differentiation protocols were time-consuming and relied heavily on growth factors, herein, we developed a three-step protocol to convert hiPSCs into DE cells in 8 days. In the first phase, hiPSCs were differentiated into non-neural ectoderm using SU5402 (an FGF signaling inhibitor). The second phase involved differentiating non-neural ectoderm into pan-placodal ectoderm and simultaneously inducing the formation of oral ectoderm (OE) using LDN193189 (a BMP signaling inhibitor) and purmorphamine (a SHH signaling activator). In the final phase, OE cells were differentiated into DE through the application of Purmorphamine, XAV939 (a WNT signaling inhibitor), and BMP4. qRT-PCR and immunostaining were performed to examine the expression of lineage-specific markers. ARS staining was performed to evaluate the formation of the mineralization nodule. The expression of PITX2, SP6, and AMBN, the emergence of mineralization nodules, and the enhanced expression of AMBN and AMELX in spheroid culture implied the generation of DE cells. This study delineates the developmental signaling pathways and uses small molecules to streamline the induction of hiPSCs into DE cells. Our findings present a simplified and quicker method for generating DE cells, contributing valuable insights for dental regeneration and dental disease research.

Dispase/collagenase cocktail allows for coisolation of satellite cells and fibroadipogenic progenitors from human skeletal muscle.

Satellite cells (SCs) and fibroadipogenic progenitors (FAPs) are progenitor populations found in muscle that form new myofibers postinjury. Muscle development, regeneration, and tissue-engineering experiments require robust progenitor populations, yet their isolation and expansion are difficult given their scarcity in muscle, limited muscle biopsy sizes in humans, and lack of methodological detail in the literature. Here, we investigated whether a dispase and collagenase type 1 and 2 cocktail could allow dual isolation of SCs and FAPs, enabling significantly increased yield from human skeletal muscle. Postdissociation, we found that single cells could be sorted into CD56 + CD31-CD45- (SC) and CD56-CD31-CD45- (FAP) cell populations, expanded in culture, and characterized for lineage-specific marker expression and differentiation capacity; we obtained ∼10% SCs and ∼40% FAPs, with yields twofold better than what is reported in current literature. SCs were PAX7+ and retained CD56 expression and myogenic fusion potential after multiple passages, expanding up to 1012 cells. Conversely, FAPs expressed CD140a and differentiated into either fibroblasts or adipocytes upon induction. This study demonstrates robust isolation of both SCs and FAPs from the same muscle sample with SC recovery more than two times higher than previously reported, which could enable translational studies for muscle injuries.NEW & NOTEWORTHY We demonstrated that a dispase/collagenase cocktail allows for simultaneous isolation of SCs and FAPs with 2× higher SC yield compared with other studies. We provide a thorough characterization of SC and FAP in vitro expansion that other studies have not reported. Following our dissociation, SCs and FAPs were able to expand by up to 1012 cells before reaching senescence and maintained differentiation capacity in vitro demonstrating their efficacy for clinical translation for muscle injury.

Joubert syndrome-derived induced pluripotent stem cells show altered neuronal differentiation in vitro.

Joubert syndrome (JS) is a recessively inherited congenital ataxia characterized by hypotonia, psychomotor delay, abnormal ocular movements, intellectual disability, and a peculiar cerebellar and brainstem malformation, the "molar tooth sign." Over 40 causative genes have been reported, all encoding for proteins implicated in the structure or functioning of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues. In this paper, we developed an in vitro neuronal differentiation model using patient-derived induced pluripotent stem cells (iPSCs), to evaluate possible neurodevelopmental defects in JS. To this end, iPSCs from four JS patients harboring mutations in distinct JS genes (AHI1, CPLANE1, TMEM67, and CC2D2A) were differentiated alongside healthy control cells to obtain mid-hindbrain precursors and cerebellar granule cells. Differentiation was monitored over 31days through the detection of lineage-specific marker expression by qRT-PCR, immunofluorescence, and transcriptomics analysis. All JS patient-derived iPSCs, regardless of the mutant gene, showed a similar impairment to differentiate into mid-hindbrain and cerebellar granule cells when compared to healthy controls. In addition, analysis of primary cilium count and morphology showed notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. These results confirm that patient-derived iPSCs are an accessible and relevant in vitro model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context.

Single-Cell Transcriptomics Reveal Altered Hematopoietic Mechanisms Driven By T21 and GATA1s

Introduction Trisomy 21 (T21) is associated with baseline erythrocytosis and thrombocytopenia and risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia of Down syndrome (ML-DS). TAM and ML-DS are characterized by mutations in the transcription factor GATA1, resulting in the truncated isoform GATA1s (G1S). We previously found that T21 increases erythropoiesis, while G1S severely impairs erythroid development but enhances megakaryocyte (MK) proliferation (Byrska-Bishop et al, JCI 2015). To better understand how T21 and G1S each impact hematopoiesis, we performed single-cell RNA sequencing (scRNA-seq) of multipotent and late hematopoietic progenitor cells (HPCs) differing only by chromosome 21 and/or G1S status. Methods Four isogenic human induced pluripotent stem cell (iPSC) lines differing only by chromosome 21 (euploid vs. T21) and/or GATA1 (WT vs. G1S) status underwent hematopoietic differentiation by embryoid body formation. Cells were collected at 3 timepoints: on day 7 (D7), CD41 +235 + multipotent HPCs were purified by flow cytometry and on days 9 (D9) and 11 (D11), late HPCs biased to a single lineage were collected. Cells were sequenced by scRNA-seq and underwent quality control followed by clustering and analysis using Seurat. Clusters were manually annotated based on expression of 141 key hematopoietic genes, including aggregated expression of lineage markers. Early vs. committed lineage clusters were determined based on the relative expression of lineage-specific markers. Within each lineage, differentially expressed genes (DEGs) associated with a genetic background were assessed using the Wilcoxon rank-sum test. The χ² test was used to compare categorical data. A 2-tailed p or adjusted p <0.05 was considered statistically significant. Results Cluster annotation revealed HPC, early and committed erythroid and MK, and myeloid cells (Figure 1). D7 T21/G1S HPCs surprisingly showed an erythroid bias, with upregulation of genes such as GYPA and AHSP, while euploid/G1S HPCs showed a MK bias, with upregulation of genes including PF4V1 and PF4. With wtGATA1, neither euploid nor T21 D7 HPCs showed a significant lineage skew. On D9 and D11, the euploid/WT, T21/WT, and T21/G1S cells developed subpopulations committing to the erythroid lineage, while the euploid/G1S cells demonstrated minimal erythroid potential (Figure 1). Across all 3 timepoints, early and committed erythrocytes from T21 and euploid cells with G1S had a greater number of combined DEGs than their wtGATA1 counterparts (G1S vs. WT, T21: 692 vs. 378; euploid: 1235 vs. 599). On D9, T21 was associated with a greater erythroid skew (38.6% of T21/WT and 32.2% of T21/G1S cells vs. 27.8% of euploid/WT cells; p <0.00001). By D11, this erythroid drive was no longer apparent, and the T21/G1S erythrocytes were significantly less mature (Table 1; p <0.00001). These data suggest that T21 initially enhances erythropoietic drive, while G1S results in a near-complete erythroid block in euploid cells but an incomplete developmental block with T21. All 4 genotypes yielded MK subpopulations on D9 and D11 (Figure 1). With wtGATA1, euploid and T21 MKs were predominantly early MKs by D11 (Table 1). Interestingly, G1S was associated with enhanced MK commitment and maturation in the euploid context but arrest at the early stage with T21 (Table 1; p <0.00001). G1S was again associated with increased DEGs compared to wtGATA1 (G1S vs. WT, T21: 594 vs. 454; euploid: 1689 vs. 1310). These data suggest that G1S in a euploid background promotes MK differentiation, but T21 and G1S cooperate to generate immature, abnormal MKs. Conclusions Although the hematopoietic abnormalities and leukemic risk of T21 have been well-described, the underlying developmental events remain unclear. This scRNA-seq timecourse of HPCs differentiated from isogenic iPSCs reveals the individual and synergistic effects of T21 and G1S. Overall, T21 enhances erythropoietic drive while G1S alone suppresses erythropoiesis. G1S in euploid cells enhances megakaryopoiesis, but with T21, megakaryopoietic commitment and maturation are uncoupled with a strong bias to early MKs. Together, the competing effects of T21 and G1S appear to result in abnormal, immature erythrocytes and MKs with impaired lineage commitment, consistent with a pro-leukemic phenotype.

Xeno-free generation of human induced pluripotent stem cells from donor-matched fibroblasts isolated from dermal and oral tissues

BackgroundInduced pluripotent stem cells (iPS) can be generated from various somatic cells and can subsequently be differentiated to multiple cell types of the body. This makes them highly promising for cellular therapy in regenerative medicine. However, to facilitate their clinical use and to ensure safety, iPS culturing protocols must be compliant with good manufacturing practice guidelines and devoid of xenogenic products. Therefore, we aimed to compare the efficiency of using humanized culture conditions, specifically human platelet lysate to fetal bovine serum, for iPS generation from different sources, and to evaluate their stemness.MethodsiPS were generated via a platelet lysate or fetal bovine serum-based culturing protocol from matched dermal, buccal and gingival human fibroblasts, isolated from healthy donors (n = 2) after informed consent, via episomal plasmid transfection. Pluripotency, genotype and phenotype of iPS, generated by both protocols, were then assessed by various methods.ResultsMore attempts were generally required to successfully reprogram xeno-free fibroblasts to iPS, as compared to xenogenic cultured fibroblasts. Furthermore, oral fibroblasts generally required more attempts for successful iPS generation as opposed to dermal fibroblasts. Morphologically, all iPS generated from fibroblasts formed tight colonies surrounded by a reflective “whitish” outer rim, typical for iPS. They also expressed pluripotency markers at both gene (SOX2, OCT4, NANOG) and protein level (SOX2, OCT4). Upon stimulation, all iPS showed ability to differentiate into the three primary germ layers via expression of lineage-specific markers for mesoderm (MESP1, OSR1, HOPX), endoderm (GATA4) and ectoderm (PAX6, RAX). Genome analysis revealed several amplifications and deletions within the chromosomes of each iPS type.ConclusionsThe xeno-free protocol had a lower reprogramming efficiency compared to the standard xenogenic protocol. The oral fibroblasts generally proved to be more difficult to reprogram than dermal fibroblasts. Xeno-free dermal, buccal and gingival fibroblasts can successfully generate iPS with a comparable genotype/phenotype to their xenogenic counterparts.

Clear cell stromal tumor of the lung with multinucleated giant cells: a report of a case with YAP1-TFE3 fusion

BackgroundClear cell (hemangioblastoma-like) stromal tumor of the lung (CCSTL) is a rare pulmonary neoplasm. Recently, 9 cases of CCSTL harboring the YAP1-TFE3 gene fusion have been described, and it has been suggested that this aberration could be a characteristic feature of this tumor.Case presentationWe here report another case of CCSTL in a 57-year-old male, which presented as a solitary lung nodule 45 mm in the greatest dimension. Microscopically, the tumor consisted of epithelioid to spindled cells with mild-to-moderate nuclear atypia, finely granular or vesicular chromatin, and small nucleoli. Nuclear indentations were a common finding. There were up to 3 mitoses per 10 HPF. The cytoplasm was slightly eosinophilic or clear. Scattered non-tumor large multinucleated cells were present. Immunohistochemically, the tumor cells showed diffuse positivity for TFE3, CD10, vimentin, and IFITM1. Other markers examined were negative, and the expression of lineage-specific markers was not found. NGS analysis revealed a fusion transcript of the YAP1 and TFE3 genes, and a pathogenic variant of the MUTYH gene.ConclusionOur finding supports the recent data suggesting that CCSTL represents a distinct entity characterized by the recurrent YAP1-TFE3 fusion.

Sustained postconfluent culture of human mammary epithelial cells enriches for luminal and c-Kit+ subtypes

BackgroundA challenge in human mammary epithelial cell (HMEC) culture is sustaining the representation of competing luminal, myoepithelial, and progenitor lineages over time. As cells replicate in culture, myoepithelial cells come to dominate the composition of the culture with serial passaging. This drift in composition presents a challenge for studying luminal and progenitor cells, which are prospective cells of origin for most breast cancer subtypes.MethodsWe demonstrate the use of postconfluent culture on HMECs. Postconfluent culture entails culturing HMECs for 2–5 weeks without passaging but maintaining frequent feedings in low-stress M87A culture medium. In contrast, standard HMEC culture entails enzymatic subculturing every 3–5 days to maintain subconfluent density.ResultsWhen compared to standard HMEC culture, postconfluent culture yields increased proportions of luminal cells and c-Kit+ progenitor cells. Postconfluent cultures develop a distinct multilayered morphology with individual cells showing decreased physical deformability as compared to cells in standard culture. Gene expression analysis of postconfluent cells shows increased expression of lineage-specific markers and extracellular matrix components.ConclusionsPostconfluent culture is a novel, useful strategy for altering the lineage composition of HMECs, by increasing the proportional representation of luminal and progenitor cells. We speculate that postconfluent culture creates a microenvironment with cellular composition closer to the physiological state and eases the isolation of scarce cell subtypes. As such, postconfluent culture is a valuable tool for researchers using HMECs for breast cancer research.

Inhibiting DNA methylation as a strategy to enhance adipose-derived stem cells differentiation: Focus on the role of Akt/mTOR and Wnt/β-catenin pathways on adipogenesis.

Adipose-derived mesenchymal stem cells (ASCs) represent a valid therapeutic option for clinical application in several diseases, due to their ability to repair damaged tissues and to mitigate the inflammatory/immune response. A better understanding of the underlying mechanisms regulating ASC biology might represent the chance to modulate their in vitro characteristics and differentiation potential for regenerative medicine purposes. Herein, we investigated the effects of the demethylating agent 5-azacytidine (5-aza) on proliferation, clonogenicity, migration, adipogenic differentiation and senescence of ASCs, to identify the molecular pathways involved. Through functional assays, we observed a detrimental effect of 5-aza on ASC self-renewal capacity and migration, accompanied by actin cytoskeleton reorganization, with decreased stress fibers. Conversely, 5-aza treatment enhanced ASC adipogenic differentiation, as assessed by lipid accumulation and expression of lineage-specific markers. We analyzed the involvement of the Akt/mTOR, MAPK and Wnt/β-catenin pathways in these processes. Our results indicated impairment of Akt and ERK phosphorylation, potentially explaining the reduced cell proliferation and migration. We observed a 5-aza-mediated inhibition of the Wnt signaling pathway, this potentially explaining the pro-adipogenic effect of the drug. Finally, 5-aza treatment significantly induced ASC senescence, through upregulation of the p53/p21 axis. Our data may have important translational implications, by helping in clarifying the potential risks and advantages of using epigenetic treatment to improve ASC characteristics for cell-based clinical approaches.

Tissue Engineering of Cartilage Using Collagen Scaffold Enriched with Plant Polysaccharides.

Degenerative diseases associated with articular cartilage pose a huge burden on health care economics. The nature of the tissue involved and the changes therein do not allow self-healing; and most of these problems are progressive. Tissue engineering offers some solutions provided we focus on the right kind of cells and the appropriate surrounding niches created for a particular tissue. The present study deals with the formation of polysaccharide rich stable scaffold of collagen after cross-linking with oxidized gum arabic. The scaffold was tested for its biocompatibility and ability to support cells. The in vitro cytotoxicity of the scaffolds toward induced pluripotent stem cells and chondrocytes was evaluated. Evaluation of expression of lineage specific markers indicates differentiation of induced pluripotent stem cells to chondrogenic lineage and maintenance of chondrocytes per se when grown in the scaffold. Animal studies were carried out to study the efficacy of the scaffold to repair the knee injuries. Cells along with the scaffold appeared to be the best filling, in repair of injured cartilage. These studies show that these scaffolds are potential candidates in applications such as tissue engineering of cartilage.

Human Dental Pulp-Derived Mesenchymal Stem Cell Potential to Differentiate into Smooth Muscle-Like Cells In Vitro.

Previous studies have shown that mesenchymal stem cells (MSCs) derived from various tissue sources can be differentiated into smooth muscle-like cells (SMLCs) in vitro. In this paper, dental pulp-derived mesenchymal stem cells (DPSCs) were evaluated for their differentiation ability towards smooth muscle-like cells (SMLCs) under the effect of widely used cytokines (TGF-β1 and PDGF-BB) with special focus on different culturing environments. For this purpose, both the commercially used culturing plates (Norm-c) and 0.1% gelatin-precoated (Gel-c) plates were used. Isolated cells displayed plastic adherence, pluripotency and cell surface marker profiling, and adipogenic and osteogenic differentiation potential with lineage specific marker expression. Differentiated cells induced under different culturing plates showed successful differentiation into SMLCs by positively expressing smooth muscle cell (SMC) specific markers both at the mRNA and protein levels. Gelatin coating could substantially enhance DPSC differentiation potential than Norm-c-induced cells. However, the absence of mature marker MHY-11 by immunostaining results from all treatment groups further indicated the development of immature and synthetic SMLCs. Finally, it was concluded that DPSC differentiation ability into SMLCs can be enhanced under cytokine treatment as well as by altering the cellular niche by precoating the culturing plates with suitable substrates. However, to get fully functional, contractile, and mature SMLCs, still many different cytokine cocktail combinations and more suitable coating substrates will be needed.

SAT-304 Pituitary Stem Cells May Drive Adenomas Causing Cushing’s Disease

Introduction:Cell rests of self-renewing Sox2+ progenitor cells have been identified in the normal pituitary glands1, however their role in human pituitary tumorigenesis is not understood. Adrenocorticotropic hormone (ACTH) producing microadenomas that cause Cushing’s disease frequently (~70%) lack pathogenic genetic mutations.2 In mice, targeted expression of oncogenic β-catenin in Sox2+ cells generate microadenomas. Interestingly, the Sox2+ cells reside within the adjacent normal gland and drive adenomas in a paracrine fashion.3 We hypothesized that Sox2+ progenitors in human pituitary gland may drive the formation of microadenomas that cause Cushing’s disease (CD). Methods:Four ACTH producing adenomas and two non-functional adenomas (NFPA) with separately annotated adjacent normal tissue (henceforward called ‘microenvironment’) were procured for this study (NCT00060541). We performed RNA deep sequencing (RNAseq) and compared expression of lineage-specific markers and progenitor markers using two-sample T-tests after testing for variance equality and using Welch’s approximation for degrees of freedom. Results:We found expected overexpression of ACTH preprohormone POMC in CD adenomas compared to adjacent microenvironment (?-fold) and NFPA (?-fold). The microenvironment in Cushing’s disease showed increased expression of progenitor markers including SOX2, SOX9, CDH1, GRFA2, and KLF4 compared with microenviroment in NFPA. Likewise, the Cushing’s disease microenvironment showed increased expression ofPOMC (26.98 - fold, P = 0.004) as well as PRLR (FC 17.39, P = 0.006) and GH1 (FC 29.91, P = 0.003) implying that increased Sox2+ progenitors contribute to terminally differentiated corticotrope, lactotroph and somatotroph lineages in-vivo. Conclusions:We report increased expression of several progenitor markers and concomitant elevation in tissues-specific markers in the microenvironment of Cushing’s disease patients. Our results indicate that increased pituitary progenitors in the microenvironment of human corticotropinomas may signal in paracrine fashion and may contribute to the pathogenesis of Cushing’s disease.

Multi-differentiation potential is necessary for optimal tenogenesis of tendon stem cells

BackgroundTendon injury is a significant clinical problem due to poor healing and a high reinjury rate; successful treatment is limited by our poor understanding of endogenous tendon stem cells. Recent evidence suggests that adult stem cells are phenotypically diverse, even when comparing stem cells isolated from the same tissue from the same individual, and may in fact exist on a spectrum of proliferation and differentiation capacities. Additionally, the relationships between and clinical relevance of this phenotypic variation are poorly understood. In particular, tenogenic capacity has not been studied in comparison to tenogenic differentiation and cell proliferation. Toward this end, we performed a comprehensive assessment of cell proliferation and differentiation capacity toward four connective tissue lineages (tendon, cartilage, bone, and adipose) using tendon stem cell lines derived from single cells released directly from tendon tissue to (1) evaluate the differences, if any, in tenogenic potential, and (2) identify the relationships between differentiation phenotypes and proliferation capacity.MethodsTendon stem cells were derived from the endotenon of superficial digital flexor tendon from 3 horses. The cell suspension from each horse was separately plated simultaneously (1) at moderate density to generate a heterogenous population of cells—parent tendon cell line—and (2) at low density to separate single cells from each other to allow isolation of colonies that derive from single mother cells—clonal tendon stem cell lines.Thirty clonal tendon stem cell lines—10 from each horse—and each parent tendon cell line were assessed for tenogenesis, tri-lineage differentiation, and cell proliferation. Differentiation was confirmed by lineage-specific cell staining and quantified by the relative gene expression of lineage-specific markers. Statistical significance was determined using analysis of variance and post hoc Tukey’s tests.ResultsThree distinct differentiation phenotypes—differentiation potency toward all 4 tissue lineages and two tri-lineage differentiation potencies—were identified in tendon clonal stem cell lines. These phenotypes were differentiation toward (1) tendon, cartilage, bone, and adipose (TCOA); (2) tendon, cartilage, and bone (TCO); and (3) tendon, cartilage, and adipose (TCA). Further, clonal cell lines that differentiated toward all four lineages had the highest expression of scleraxis and mohawk upon tenogenesis. Moreover, cell proliferation was significantly different between phenotypic groups, as evidenced by increased numbers of cumulative cell population doublings in clonal cell lines that did not differentiate toward adipose.ConclusionsOur study provides evidence of the heterogenous character of adult stem cells and identifies key differences in tendon stem cell differentiation and proliferative potentials from the same individual and from the same tendon. Isolation of tendon stem cell lines with the capacity to differentiate into all four connective tissue lineages may yield improved therapeutic benefits in clinical models of repair and promote a native, regenerative phenotype in engineered tendons. Future studies may be targeted to understanding the functional contributions of each tendon stem cell phenotype in vivo and identifying additional cell phenotypes.

SOX2 protein transduction directly converts human fibroblasts into oligodendrocyte-like cells

Oligodendrocyte precursor cells (OPCs) are ideal therapeutic cells for treatment of spinal cord injuries and diseases that affect myelin. However, it is necessary to generate a cell population with a low risk of teratoma formation and oncogenesis from a patient’s somatic cells. In this study, we investigated the direct reprogramming of fibroblasts to oligodendrocyte-like cells in one step with a safe non-genetic delivery method that used protein transduction. Cell morphology and the lineage-specific marker expression profile indicated that human foreskin fibroblasts (HFFs) were converted into oligodendrocyte-like cells by the application of pluripotency factors and the use of a permissible induction medium. Our data demonstrated that SOX2 was sufficient to directly drive OPC fate conversion from HFF by a genetic-free approach. Therefore, this work has provided a strategy to OPC reprogramming by a non-integrating approach for future use in disease modeling and may ultimately provide applications for patient-specific cell-based regenerative medicine.

Glycogen synthase kinase 3β inhibitor- CHIR 99021 augments the differentiation potential of mesenchymal stem cells

AimMesenchymal stem cells (MSCs) are immunomodulatory, non-teratogenic and multipotent alternatives to embryonic or induced pluripotent stem cells (ESCs or iPSCs). However, the potency of MSCs is not equivalent to the pluripotency of ESCs or iPSCs. We used CHIR 99021 to improve current protocols and methods of differentiation for the enhanced transdifferentiation potency of MSCs. Main MethodsWe used Flurescence activated cell sorter (FACS) for MSC immunophenotyping and biochemical assay for demonstrating the trilineage potential of MSCs. We used real-time polymerase chain reaction, immunocytochemistry and Western blotting assay for analyzing the expression of lineage-specific markers. Key FindingsCHIR 99021 treatment of MSCs resulted in enhanced transdifferentiation into neurological, hepatogenic and cardiomyocyte lineages with standardized protocols of differentiation. CHIR 99021–treated MSCs showed increased nuclear localization of β-catenin. These MSCs showed a significantly increased deposition of active histone marks (H3K4Me3, H3K36Me3), whereas no change was observed in repressive marks (H3K9Me3, H3K27Me3). Differential methylation profiling showed demethylation of the transcription factor OCT4 promoter region with subsequent analysis revealing increased gene expression and protein content. The HLA-DR antigen was absent in CHIR 99021–treated MSCs and their differentiated cell types, indicating their immune-privileged status. Karyotyping analysis showed that CHIR 99021–treated MSCs were genomically stable. Teratoma analysis of nude mice injected with CHIR 99021–treated MSCs showed the increased presence of cell types of mesodermal origin at the site of injection. SignificanceMSCs pretreated with CHIR 99021 can be potent, abundant alternative sources of stem cells with enhanced differentiation capabilities that are well suited to cell-based regenerative therapy.

Gene Expression Patterns of Royan Human Embryonic Stem Cells Correlate with Their Propensity and Culture Systems.

Objective Human embryonic stem cells (hESCs) have the potential to give rise to all types of cells in the human body when appropriately induced to differentiate. Stem cells can differentiate spontaneously into the three-germ layer derivatives by embryoid bodies (EBs) formation. However, the two-dimensional (2D) adherent culture of hESCs under defined conditions is commonly used for directed differentiation toward a specific type of mature cells. In this study, we aimed to determine the propensity of the Royan hESC lines based on comparison of expression levels of 46 lineage specific markers. Materials and Methods In this experimental study, we have compared the expression of lineage-specific markers in hESC lines during EB versus adherent-based spontaneous differentiation. We used quantitative real-time polymerase chain reaction (qRT-PCR) to assess expressions of 46 lineage-specific markers in 4 hESC lines, Royan H1 (RH1), RH2, RH5, and RH6, during spontaneous differentiation in both EB and adherent cultures at 0, 10, and 30 days after initiation of differentiation. Results Based on qRT-PCR data analysis, the liver and neuronal markers had higher expression levels in EBs, whereas skin-specific markers expressed at higher levels in the adherent culture. The results showed differential expression patterns of some lineage-specific markers in EBs compared with the adherent cultures. ConclusionAccording to these results, possibly the spontaneous differentiation technique could be a useful method for optimization of culture conditions to differentiate stem cells into specific cell types such ectoderm, neuron, endoderm and hepatocyte. This approach might prove beneficial for further work on maximizing the efficiency of directed differentiation and development of novel differentiation protocols.

Polyurethane/Hydroxyapatite Induces MSCs towards Osteo-like Cells in a Similar Fashion to Demineralized Bone Matrix

Osteoarthritis (OA) is the single most prevalent disorder in older adults having a predicted value of 130 million patients in 2050. Several clinical chemotherapeutic approaches are being applied to treat early or late osteoarthritis. It has been recommended that autologous mesenchymal stem cells (MSCs) from OA patients could be the gold standard to treat OA as these cells have high proliferation and chondrocyte lineage differentiation potential. In this work, human MSCs, derived from adipose tissue (Ad-MSCs), loaded on Polyurethane/Hydroxyapatite (PUHA) and Demineralized Bone Matrix (DBM) and their proliferation, differentiation capabilities were determined by MTT assay and Alizarin Red S staining and the expression of mRNA into osteoblast lineage were determined using Real Time PCR . The result showed that MSCs were more viable on PUHA when compared with DBM and the expression of lineage specific markers showed that differentiation potential of PUHA and DBM was not much different. The osteoblast lineage cells were stained positively with Alizarin Red S in completely similar in both groups. Electron microscopy analysis indicated attachment of Ad-MSCs when cultured on the PUHA and DBM. It was concluded that PUHA can be used in clinics as Osteo-inductive scaffold to treat OA easily, however further investigations are required before moving to clinical studies.

Lung non-terminal respiratory unit type adenocarcinoma: a clinicopathologic study

Objective: To evaluate the clinicopathologic characteristics of lung non-terminal respiratory unit (non-TRU) type adenocarcinoma. Methods: Seventy-two cases of lung non-TRU type adenocarcinoma that underwent complete resection and diagnosed at Departments of Pathology, Affiliated Suzhou Hospital of Nanjing Medical University and Nanjing General Hospital of the PLA from January 2005 to December 2016 were retrospectively studied. The histomorphological changes and precursor lesions were observed under microscope. The expression of lineage-specific markers and tumor stem cell markers was detected by immunohistochemistry (IHC). The major driver mutations of lung adenocarcinoma were tested by ARMS and directive gene sequencing. Results: Non-TRU type adenocarcinomas were more commonly found in male (65.3%, 47/72), former or current smokers (68.1%, 49/72), the elder (mean 61 years old), central adenocarcinoma (75.0%, 54/72), tumors with necrosis (61.1%, 44/72) and higher grade (73.6%, 53/72). Histologically, non-TRU type adenocarcinoma displayed complex histomorphology and was often composed of large irregular gland-like and acinar pattern accumulating extracellular mucin, necrotic tumor cell debris and neutrophils, or invasive adenocarcinoma with mucin production. The tumor cells were composed of bronchial surface epithelial cells, mucinous column cells, polygonal cells and goblet cells. Eighteen (25.0%), 23 (31.9%) and 28 (38.9%) cases exhibited ciliated columnar cell metaplasia (CCCM), mucous columnar cell change (MCCC) and bronchiolar columnar cell dysplasia (BCCD) (precursor lesion of lung adenocarcinoma). IHC showed the expression of CK7 (100.0%, 72/72), TTF1 (12.5%, 9/72), Napsin A (5.6%, 4/72), MUC5AC (81.9%, 59/72), MUC5B (87.5%, 63/72), p53 (66.7%, 48/72), CK5/6 (12.5%, 9/72), p63 (18.1%, 13/72), CK20 (19.4%, 14/72) and CDX2 (16.7%, 12/72) in the tumor cells. The expression of tumor stem cell markers was detected in 43.1% cases (31/72) for CD44, 31.9% (23/72) for CD133, 58.3% (42/72) for β-catenin, 36.1% (26/72) for ALDH1, 12.5% (9/72) for GATA6, 20.8% (15/72) for SOX2 and 29.2% (21/72) for OCT4. The driver mutations were 26.4% (19/72) for KRAS, 2.8% (2/72) for EGFR and 1.4% (1/72) for EML4-ALK, and none for BRAF and ROS1. Conclusion: Non-TRU type adenocarcinoma is an uncommon subtype of lung adenocarcinoma with distinct clinicopathologic characteristics, histologic appearances, immunophenotype and molecular genetic alterations.