Differentiation Potential Of Cells Research Articles

SALL3 mediates the loss of neuroectodermal differentiation potential in human embryonic stem cells with chromosome 18q loss

Human pluripotent stem cell (hPSC) cultures are prone to genetic drift, because cells that have acquired specific genetic abnormalities experience a selective advantage invitro. These abnormalities are highly recurrent in hPSC lines worldwide, but their functional consequences in differentiating cells are scarcely described. In this work, we show that the loss of chromosome 18q impairs neuroectoderm commitment and that downregulation of SALL3, a gene located in the common 18q loss region, is responsible for this failed neuroectodermal differentiation. Knockdown of SALL3 in control lines impaired differentiation in a manner similar to the loss of 18q, and transgenic overexpression of SALL3 in hESCs with 18q loss rescued the differentiation capacity of the cells. Finally, we show that loss of 18q and downregulation of SALL3 leads to changes in the expression of genes involved in pathways regulating pluripotency and differentiation, suggesting that these cells are in an altered state of pluripotency.

Topological and geometric analysis of cell states in single-cell transcriptomic data.

Single-cell RNA sequencing (scRNA-seq) enables dissecting cellular heterogeneity in tissues, resulting in numerous biological discoveries. Various computational methods have been devised to delineate cell types by clustering scRNA-seq data, where clusters are often annotated using prior knowledge of marker genes. In addition to identifying pure cell types, several methods have been developed to identify cells undergoing state transitions, which often rely on prior clustering results. The present computational approaches predominantly investigate the local and first-order structures of scRNA-seq data using graph representations, while scRNA-seq data frequently display complex high-dimensional structures. Here, we introduce scGeom, a tool that exploits the multiscale and multidimensional structures in scRNA-seq data by analyzing the geometry and topology through curvature and persistent homology of both cell and gene networks. We demonstrate the utility of these structural features to reflect biological properties and functions in several applications, where we show that curvatures and topological signatures of cell and gene networks can help indicate transition cells and the differentiation potential of cells. We also illustrate that structural characteristics can improve the classification of cell types.

Abstract 264: Differentiation therapy: Esculetin as a potent agent to alter cellular plasticity of leukemic & solid tumor stem cells

Abstract Background The “Differentiation therapy” has been emerging as a promising and more effective strategy against acute leukemia relapses. Objective In extension to the revolutionising therapeutic outcomes of All Trans Retinoic Acid (ATRA) to induce terminal differentiation of Acute Promyelocytic Leukemic (APL) blast cells, we decipher the potential effect of a natural compound “Esculetin” to serve as a differentiating agent in Acute Myeloid Leukemia (AML) as well as solid tumour cells. Underlaying role of Wnt signaling pathways in esculetin mediated blast cell differentiation was also evaluated. Methods Human acute myeloid leukemic cells (Kasumi-1) with t(8;21/AML-ETO) translocation were used as a model system. Growth inhibitory and cytotoxic activity of esculetin were analysed using growth kinetics and MTT assay. Morphological alterations, cell scatter characteristics, NBT reduction assay and cell surface marker expression pat- terns were analysed to detect terminally differentiated phenotypes. We employed RT2profiler PCR array system for the analysis of transcriptome profile of Wnt signaling components. Calcium inhibitors (TMB8 and Amlodipine) and Transforming growth factor beta (TGF-β) were used to modulate the Wnt signaling axes. To investigate whether esculetin exerts the similar effects on the invasive solid tumour cancer stem cells subset, we used a cellular model system of colon carcinoma HCT116 cells reflecting EMT phenotype. Results We illustrate cytotoxic as well as blast cell differentiation potential of esculetin on Kasumi-1 cells. Morphological alterations akin to neutrophilic differentiation as well as the corresponding acquisition of myeloid lineage markers indicate terminal differentiation potential of esculetin in leukemic blast cells. Exposure to esculetin also resulted in downregulation of canonical Wnt axis while upto ~ 21 fold upregulation of non-canonical axis associated genes. Esculetin also showed potential to revert the CSC marker expressions and concurrent EMT consistent with reduced functional aggressiveness in colon cancer cells. Conclusions Our study highlights the importance of selective use of calcium pools as well as “axis shift” of the canonical to non-canonical Wnt signaling upon esculetin treatment which might abrogate the inherent proliferation to release maturation arrest and induce the differentiation in leukemic blast cells as well as CSC stem cells. The current findings provide further therapeutic interventions to consider esculetin as a potent differentiating agent to counteract relapses. Citation Format: Ankit Mathur, Daman Saluja. Differentiation therapy: Esculetin as a potent agent to alter cellular plasticity of leukemic & solid tumor stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 264.

Autophagy Inhibition-induced Cytosolic DNA Sensing Combined with Differentiation Therapy Induces Irreversible Myeloid Differentiation in Leukemia Cells.

Clinical effects on AML therapy are closely associated with reactivating the normal myeloid differentiation potential in leukemia cells. This study shows that autophagosome formation inhibitors activate the cytosolic DNA-sensor signaling, thereby augmenting conventional differentiation therapy to induce irreversible differentiation and cell growth arrest in several types of AML cell lines.

C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription

BackgroundAcute myeloid leukemia (AML) with biallelic (CEBPAbi) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately, leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs.MethodsHuman AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis, ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally, an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents.ResultsWe found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA, DDIT3 expression was upregulated, resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3, thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However, C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia, and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally, we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers, such as tunicamycin and sorafenib in vitro and in vivo.ConclusionOur results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g., CEBPAbi) may not benefit from monotherapy with BCL2 inhibitors. However, this issue can be resolved by combining ER stress inducers.

Harnessing Mechanical Stress with Viscoelastic Biomaterials for Periodontal Ligament Regeneration.

The viscoelasticity of mechanically sensitive tissues such as periodontal ligaments (PDLs) is key in maintaining mechanical homeostasis. Unfortunately, PDLs easily lose viscoelasticity (e.g., stress relaxation) during periodontitis or dental trauma, which disrupt cell-extracellular matrix (ECM) interactions and accelerates tissue damage. Here, Pluronic F127 diacrylate (F127DA) hydrogels with PDL-matched stress relaxation rates and high elastic moduli are developed. The hydrogel viscoelasticity is modulated without chemical cross-linking by controlling precursor concentrations. Under cytomechanical loading, F127DA hydrogels with fast relaxation rates significantly improved the fibrogenic differentiation potential of PDL stem cells (PDLSCs), while cells cultured on F127DA hydrogels with various stress relaxation rates exhibited similar fibrogenic differentiation potentials with limited cell spreading and traction forces under static conditions. Mechanically, faster-relaxing F127DA hydrogels leveraged cytomechanical loading to activate PDLSC mechanotransduction by upregulating integrin-focal adhesion kinase pathway and thus cytoskeletal rearrangement, reinforcing cell-ECM interactions. In vivo experiments confirm that faster-relaxing F127DA hydrogels significantly promoted PDL repair and reduced abnormal healing (e.g., root resorption and ankyloses) in delayed replantation of avulsed teeth. This study firstly investigated how matrix nonlinear viscoelasticity influences the fibrogenesis of PDLSCs under mechanical stimuli, and it reveals the underlying mechanobiology, which suggests novel strategies for PDL regeneration.

Mouse mandibular-derived osteoclast progenitors have differences in intrinsic properties compared with femoral-derived progenitors.

Craniofacial osteoclasts are essential for site-specific processes such as alveolar bone resorption, tooth eruption, and orthodontic tooth movement. Much of the current understanding of osteoclast development and function comes from studies using long bone-derived cells. Minimal investigation has been done to explore skeletal site differences. The overall goal of this study was to determine if mandibular- and femoral-derived osteoclasts represent distinct populations. To test this hypothesis, bone marrow cells were initially analyzed from the mandible and femur of 2-month-old mice. It was shown that mandibular-derived osteoclasts have enhanced size (mm2) compared with femoral-derived osteoclasts. Since bone marrow macrophages are a heterogenous population, we additionally selected for monocytes and demonstrated that mandibular-derived monocytes also form osteoclasts with increased size compared with femoral-derived monocytes. Osteoclast precursor populations from both skeletal sites were analyzed by flow cytometry. A newly described Ly6CHigh+ population as well as the Ly6Cint population was increased in the mandibular-derived cells. The difference in differentiation potential between monocyte cultures suggests that the increase in the Ly6CHigh+ population may explain the enhanced differentiation potential in mandibular-derived cells. Monocyte genes such as Pu.1, C/ebp-a, and Prdm1 are increased in expression in mandibular-derived monocytes compared with femoral-derived monocytes. As expected with enhanced differentiation, osteoclast genes including Nfatc1, Dc-stamp, Ctsk, and Rank are upregulated in mandibular-derived osteoclast precursors. Future studies will determine how changes in the environment of the mandible lead to changes in percentages of osteoclast progenitors and their differentiation potential.

Investigation of the differentiation potential of pericyte cells as an alternative source of mesenchymal stem cells

BackgroundThe mesenchymal stem cells (MSCs) with characterized by their multipotency and capacity to differentiate into various tissue cell types, have led to their incorporation in regenerative medicine research. However, the limited numbers of MSCs in the human body and their diverse differentiation capabilities in tissues highlight the need for exploring alternative regenerative cell sources. In this study, therefore, we conducted molecular level examinations to determine whether pericytes, specialized cell communities situated near blood vessels, could serve as a substitute for human bone marrow-derived mesenchymal stem cells (hBM-MSCs). In this context, the potential application of pericytes surrounds the vessels when MSCs are insufficient for functional purposes. MethodsThe pericytes utilized in this investigation were derived from the placenta and characterized at the third passage. Similarly, the hBM-MSCs were also characterized at the third passage. The pluripotent properties of the two cell types were assessed at the gene expression level. Thereafter, both pericytes and hBM-MSCs were directed towards adipogenic, osteogenic and chondrogenic differentiation. The cells in both groups were examined on days 7, 14, and, 21 and their differentiation status was compared both immunohistochemically and through gene expression analysis. ResultsUpon comparing the pluripotency characteristics of placental pericytes and hBM-MSCs, it was discovered that there was a substantial upregulation of the pluripotency genes FoxD3, Sox2, ZPF42, UTF1, and, Lin28 in both cell types. However, no significant expression of the genes Msx1, Nr6a1, Pdx1, and, GATA6 was observed in either cell type. It was also noted that pericytes differentiate into adipogenic, osteogenic and, chondrogenic lineages similar to hBM-MSCs. DiscussionAs a result, it has been determined that pericytes exhibit high differentiation and proliferation properties similar to those of MSCs, and therefore can be considered a suitable alternative cell source for regenerative medicine and tissue engineering research, in cases where MSCs are not available or insufficient. It is notable that pericytes have been suggested as a potential substitute in studies where MSCs are lacking.

Tonic TCR and IL-1β signaling mediate phenotypic alterations of naive CD4+ T cells

Inert naive CD4+ T (TN) cells differentiate into functional T helper (Th) or regulatory T (Treg) cell subsets upon encountering antigens, mediating properly directed immune responses. Although all TN cells can differentiate into any of the Th and Treg cell subsets, heterogeneity exists among TN cells. By constructing reporter mice to detect ongoing Tcell receptor (TCR) signaling, we identify that interleukin (IL)-1β signaling affects TN cell characteristics, independent of tonic TCR signaling, which also alters TN cell phenotypes. IL-1β reversibly attenuates the differentiation potential of TN cells toward Treg cells. IL-1β signaling is elevated in the splenic TN cells, consequently attenuating their differentiation potential toward Treg cells. Aberrant elevation of IL-1β signaling augments colitogenic activities of TN cells. TN cells in patients with colitis exhibited elevated IL-1β signaling. We demonstrate that phenotypic alteration in TN cells by IL-1β is an important mechanism in the regulation of immune responses.

Fibrochondrogenic Differentiation Potential of Human Adiposederived Mesenchymal Stem Cells in a Type I Collagen-based Meniscus Scaffold with Activated Platelet-Rich Plasma Stimulation In-vitro

Introduction: Despite efforts to use scaffolds to treat meniscus tears, minimal progress has been made in facilitating meniscus regeneration and return of function. Our research objective was to develop a meniscus repair and regeneration implant by applying a resorbable scaffold in combination with cells and growth factors. We report here the results of using Platelet-Rich Plasma (PRP) as a source of growth factors to induce fibrochondrogenic differentiation of human Adipose- Derived Mesenchymal Stem Cells (hADSC) in a three-dimensional (3D) Type I collagen-based scaffold in-vitro. Methods: Scaffold Preparation: Type I collagen scaffolds were prepared following a protocol previously published. Two different densities of scaffolds, High Density (HD) and Low Density (LD), were produced for in-vitro study. hADSC and PRP Preparation. hADSCs were cultured to the fifth passage to reach the desired number for experimentation. PRP was collected from human blood and activated. Cell Culture Procedure: Effects of PRP on hADSC proliferation and differentiation into fibrochondrogenic cells were examined in four scaffold groups: LD, HD, LD+PRP and HD+PRP. hADSCs were seeded onto scaffolds (n=5) at a concentration of 2 × 106 cells/scaffold. 1% of PRP was added to the experimental media. Cellular proliferation was assessed at 1, 7, 14 and 21 days. Differentiation was measured using qRT-PCR on Days 14 and 21. qRT- PCR analysis of gene expression was completed with primers for COLLAGEN 1 and AGGRECAN. Data Analysis: ANOVAs were conducted (two-tailed tests) at the .05 significance level. Results: Cellular proliferation of hADSCs seeded on each scaffold increased over time. Similar trend was observed for cells seeded on HD scaffolds with and without PRP. hADSC showed significant increase in cellular proliferation on the LD scaffolds at Days 1 and 7. At Day 21, PRP treatment and LD scaffold had a synergistic positive effect on Type I collagen gene expression. PRP did not elevate type I collagen gene in the HD group, the HD scaffold alone had the same level of type I collagen gene expression as LD+PRP. Aggrecan expression was elevated in the presence of PRP in both the HD and LD scaffold groups, indicating enhanced fibrochondrogenic differentiation of hADSCs. Effective cell infiltration was observed across both HD and LD scaffolds with and without PRP treatment. HD scaffolds displayed larger cell clusters and more extensive cell migration over time compared to LD scaffolds. However, LD scaffolds resulted a more uniform cellular distribution than HD scaffolds. Conclusion: Our study demonstrates that PRP can play an important role in directing hADSCs towards fibrochondrogenic differentiation in Type I collagen-based scaffolds in-vitro. Additionally, our study shows that collagen scaffold density can influence the spatial distribution and cellular behavior of infiltrated cells.

A Reversible Neural Stem Cell Quiescence and Activation Culture System for Metabolic Study.

Stem cells in vivo can transit between quiescence and activation, two metabolically distinct states. It is increasingly appreciated that cell metabolism assumes profound roles in stem cell maintenance and tissue homeostasis. However, the lack of suitable models greatly hinders our understanding of the metabolic control of stem cell quiescence and activation. In the present study, we have utilized classical signaling pathways and developed a cell culture system to model reversible NSC quiescence and activation. Unlike activated ones, quiescent NSCs manifested distinct morphology characteristics, cell proliferation, and cell cycle properties but retained the same cell proliferation and differentiation potentials once reactivated. Further transcriptomic analysis revealed that extensive metabolic differences existed between quiescent and activated NSCs. Subsequent experimentations confirmed that NSC quiescence and activation transition was accompanied by a dramatic yet coordinated and dynamic shift in RNA metabolism, protein synthesis, and mitochondrial and autophagy activity. The present work not only showcases the broad utilities of this powerful in vitro NSC quiescence and activation culture system but also provides timely insights for the field and warrants further investigations.

3D bioprinted small extracellular vesicles from periodontal cells enhance mesenchymal stromal cell function

Recent research indicates that combining 3D bioprinting and small extracellular vesicles (sEVs) offers a promising ‘cell-free’ regenerative medicine approach for various tissue engineering applications. Nonetheless, the majority of existing research has focused on bioprinting of sEVs sourced from cell lines. There remains a notable gap in research regarding the bioprinting of sEVs derived from primary human periodontal cells and their potential impact on ligamentous and osteogenic differentiation. Here, we investigated the effect of 3D bioprinted periodontal cell sEVs constructs on the differentiation potential of human buccal fat pad-derived mesenchymal stromal cells (hBFP-MSCs). Periodontal cell-derived sEVs were enriched by size exclusion chromatography (SEC) with particle-shaped morphology, and characterized by being smaller than 200 nm in size and CD9/CD63/CD81 positive, from primary human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs). The sEVs were then 3D bioprinted in 10 % gelatin methacryloyl (GelMA) via microextrusion bioprinting. Release of sEVs from bioprinted constructs was determined by DiO-labelling and confocal imaging, and CD9 ELISA. Attachment and ligament/osteogenic/cementogenic differentiation of hBFP-MSCs was assessed on bioprinted GelMA, without and with sEVs (GelMA/hPDLCs-sEVs and GelMA/hGFs-sEVs), scaffolds. hBFP-MSCs seeded on the bioprinted sEVs constructs spread well with significantly enhanced focal adhesion, mechanotransduction associated gene expression, and ligament and osteogenesis/cementogenesis differentiation markers in GelMA/hPDLCs-sEVs, compared to GelMA/hGFs-sEVs and GelMA groups. A 2-week osteogenic and ligamentous differentiation showed enhanced ALP staining, calcium formation and toluidine blue stained cells in hBFP-MSCs on bioprinted GelMA/hPDLCs-sEVs constructs compared to the other two groups. The proof-of-concept data from this study supports the notion that 3D bioprinted GelMA/hPDLCs-sEVs scaffolds promote cell attachment, as well as ligamentous, osteogenic and cementogenic differentiation, of hBFP-MSCs in vitro.

Characteristics of the synovial microenvironment and synovial mesenchymal stem cells with hip osteoarthritis of different bone morphologies

BackgroundVariations in bone morphology in patients with hip osteoarthritis (HOA) can be broadly categorized into three types: atrophic, normotrophic, and hypertrophic. Despite the investigations examining clinical elements, such as bone morphology, pain, and range of motion, our understanding of the pathogenesis of HOA remains limited. Previous studies have suggested that osteophytes typically originate at the interface of the joint cartilage, periosteum, and synovium, potentially implicating synovial mesenchymal stem cells (SMSCs) in the process. This study aimed to investigate the potential factors that drive the development of bone morphological features in HOA by investigating the characteristics of the synovium, differentiation potential of SMSCs, and composition of synovial fluid in different types of HOA.MethodsSynovial tissue and fluid were collected from 30 patients who underwent total hip arthroplasty (THA) with the variable bone morphology of HOA patients. RNA sequencing analysis and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were performed to analyse the genes in the normotrophic and hypertrophic synovial tissue. SMSCs were isolated and cultured from the normotrophic and hypertrophic synovial tissues of each hip joint in accordance with the variable bone morphology of HOA patients. Cell differentiation potential was compared using differentiation and colony-forming unit assays. Cytokine array was performed to analyse the protein expression in the synovial fluid.ResultsIn the RNA sequencing analysis, 103 differentially expressed genes (DEGs) were identified, predominantly related to the interleukin 17 (IL-17) signalling pathway. Using a protein–protein interaction (PPI) network, 20 hub genes were identified, including MYC, CXCL8, ATF3, NR4A1, ZC3H12A, NR4A2, FOSB, and FOSL1. Among these hub genes, four belonged to the AP-1 family. There were no significant differences in the tri-lineage differentiation potential and colony-forming capacity of SMSCs. However, RT-qPCR revealed elevated SOX9 expression levels in synovial tissues from the hypertrophic group. The cytokine array demonstrated significantly higher levels of CXCL8, MMP9, and VEGF in the synovial fluid of the hypertrophic group than in the normotrophic group, with CXCL8 and MMP9 being significantly expressed in the hypertrophic synovium.ConclusionUpregulation of AP-1 family genes in the synovium and increased concentrations of CXCL8, MMP9, and VEGF were detected in the synovial fluid of the hypertrophic group of HOA patients, potentially stimulating the differentiation of SMSCs towards the cartilage and thereby contributing to severe osteophyte formation.

A comprehensive stem cell laboratory module with blended learning for medical students at Tongji University.

The laboratory practice "Primary culture and directional differentiation of rat bone marrow mesenchymal stem cells (BMSCs)" is part of a required course for sophomore medical students at Tongji university, which has been conducted since 2012. Blended learning has been widely applied in medical courses. Based on a student-centered teaching philosophy, we reconstructed a comprehensive stem cell laboratory module with blended learning in 2021, aiming to facilitate students in enhancing their understanding of the multi-lineage differentiation potential of stem cells and improve their experimental skills, self-directed learning ability, and innovative thinking. First, we constructed in-depth online study resources, including videos demonstrating laboratory procedures, a PowerPoint slide deck, and published literature on student self-learning before class. In class, students performed a primary culture of BMSCs, freely chose among adipogenic, osteogenic, or chondrogenic differentiation, and used cytochemical or immunofluorescence staining for identification. After class, the extracurricular part involved performing quantitative polymerase chain reaction to examine the expression of multi-lineage differentiation marker genes, which was designed as an elective. After 2 years of practice, positive feedback was obtained from both students and faculty members who achieved, the learning goal as expected. The reconstructed stem cell laboratory module provides comprehensive practice opportunities for students. Students have a better understanding of BMSC at the molecular, cellular, and functional levels and have improved their experimental skills, which forms a basis for scientific research for medical students. Introducing blended learning into other medical laboratory practices thus seems valuable.

Generation of a human induced pluripotent stem cell line overexpressing CCL22 with islet cells differentiation potential

CCL22 is a macrophage-derived immunosuppressive chemokine that recruits regulatory T cells through the CCL22:CCR4 axis, playing an important role in homeostatic and inflammatory responses. A CCL22-overexpressing human induced pluripotent stem cell line (CNNDi001-A-2) was generated by lentiviral transduction to further study the function of CCL22. The cell line was confirmed to have normal proliferation and pluripotency and could be further differentiated into islet cells for cell replacement therapy in diabetes.

Proliferation and Differentiation Potential of Bone Marrow-Derived Mesenchymal Stem Cells From Children With Polydactyly and Adults With Basal Joint Arthritis.

This study compared the proliferation and differentiation potential of bone marrow-derived mesenchymal stem cells (BMSCs) derived from infants with polydactyly and adults with basal joint arthritis. The proliferation rate of adult and infant BMSCs was determined by the cell number changes and doubling times. The γH2AX immunofluorescence staining, age-related gene expression, senescence-associated β-galactosidase (SA-β-gal) staining were analyzed to determine the senescence state of adult and infant BMSCs. The expression levels of superoxide dismutases (SODs) and genes associated with various types of differentiation were measured using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Differentiation levels were evaluated through histochemical and immunohistochemical staining. The results showed that infant BMSCs had a significantly higher increase in cell numbers and faster doubling times compared with adult BMSCs. Infant BMSCs at late stages exhibited reduced γH2AX expression and SA-β-gal staining, indicating lower levels of senescence. The expression levels of senescence-related genes (p16, p21, and p53) in infant BMSCs were also lower than in adult BMSCs. In addition, infant BMSCs demonstrated higher antioxidative ability with elevated expression of SOD1, SOD2, and SOD3 compared with adult BMSCs. In terms of differentiation potential, infant BMSCs outperformed adult BMSCs in chondrogenesis, as indicated by higher expression levels of chondrogenic genes (SOX9, COL2, and COL10) and positive immunohistochemical staining. Moreover, differentiated cells derived from infant BMSCs exhibited significantly higher expression levels of osteogenic, tenogenic, hepatogenic, and neurogenic genes compared with those derived from adult BMSCs. Histochemical and immunofluorescence staining confirmed these findings. However, adult BMSCs showed lower adipogenic differentiation potential compared with infant BMSCs. Overall, infant BMSCs demonstrated superior characteristics, including higher proliferation rates, enhanced antioxidative activity, and greater differentiation potential into various lineages. They also exhibited reduced cellular senescence. These findings, within the context of cellular differentiation, suggest potential implications for the use of allogeneic BMSC transplantation, emphasizing the need for further in vivo investigation.

Differentiation potential of human amniotic fluid-derived mesenchymal stem cells into hepatocyte-like cells

Differentiation potential of human amniotic fluid-derived mesenchymal stem cells into hepatocyte-like cells

Sex hormone-binding globulin promotes the osteogenic differentiation potential of equine adipose-derived stromal cells by activating the BMP signaling pathway.

Musculoskeletal injuries and chronic degenerative diseases pose significant challenges in equine health, impacting performance and overall well-being. Sex Hormone-Binding Globulin (SHBG) is a glycoprotein determining the bioavailability of sex hormones in the bloodstream, and exerting critical metabolic functions, thus impacting the homeostasis of many tissues including the bone. In this study, we investigated the potential role of SHBG in promoting osteogenesis and its underlying mechanisms in a model of equine adipose-derived stromal cells (ASCs). An SHBG-knocked down model has been established using predesigned siRNA, and cells subjected to osteogenic induction medium in the presence of exogenous SHBG protein. Changes in differentiation events where then screened using various analytical methods. We demonstrated that SHBG treatment enhances the expression of key osteoconductive regulators in equine ASCs CD34+ cells, suggesting its therapeutic potential for bone regeneration. Specifically, SHBG increased the cellular expression of BMP2/4, osteocalcin (OCL), alkaline phosphatase (ALP), and osteopontin (OPN), crucial factors in early osteogenesis. Furthermore, SHBG treatment maintained adequate apoptosis and enhanced autophagy during osteogenic differentiation, contributing to bone formation and remodeling. SHBG further targeted mitochondrial dynamics, and promoted the reorganization of the mitochondrial network, as well as the expression of dynamics mediators including PINK, PARKIN and MFN1, suggesting its role in adapting cells to the osteogenic milieu, with implications for osteoblast maturation and differentiation. Overall, our findings provide novel insights into SHBG's role in bone formation and suggest its potential therapeutic utility for bone regeneration in equine medicine.

The Differentiation Potential of Apical Papilla Cells in Relation to Tenascin-C and Syndecan-1 Expression and Their Potential Role in Regeneration.

Introduction: This study investigated the distribution pattern of tenascin-C and syndecan-1 in the dental mesenchyme during root development of immature swine teeth in order to define the differentiation dynamics of both pulp tissue progenitors and apical papilla cells, as well as to assess the adequacy criticize of the apical papilla to induce dentin-pulp regeneration. Methods: Three 7-month-old miniature swine were used in this study. A total of 12 teeth, including two immature permanent incisors and two premolar teeth of each case, were extracted and processed for histological and immunohistochemical analysis. Different populations of mesenchymal cells located at the root apex were morphologically evaluated in hematoxylin-eosin serial sections. Additionally, the distribution patterns of tenascin-C and syndecan-1 were assessed immunohistochemically. Results: Syndecan-1 was strongly expressed in the dental pulp, particularly along the odontoblasts of the root and the newly deposited predentin layer. Tenascin-C was intensely expressed in the dental pulp. The apical papilla and dental follicle showed no expression of either molecule. Conclusions: Cell differentiation potential in the developing swine apex is progressively restricted to the newly formed dental pulp, whereas phenotypic expression of apical papilla cells remains undetermined unless the new microenvironment triggers cell differentiation towards the odontoblastic lineage.

Development of an intestinal epithelial cell line and organoids derived from the same swine and characterization of their antiviral responses.

Intestinal homeostasis and integrity are important factors for maintaining host health. This study established intestinal epithelial cell lines and organoids from the same swine jejunal crypts to develop seamless swine intestinal in vitro evaluation systems. The study evaluated the proliferative capacity and tight junction formation of the epithelial cell line and characterized the cell differentiation potential of the intestinal organoids. The evaluation systems were subsequently exposed to the Toll-like receptor 3 (TLR3) agonist poly(I:C) to simulate viral infections and assess the antiviral responses. The results demonstrated no differences in the response to type I interferons. There were, however, significant differences in the expression of interferon-stimulated genes. This study collectively introduced a flexible evaluation system using cell lines and organoids and revealed notable differences in the expression of interferon-stimulated genes, highlighting the complexity of the immune responses in these in vitro systems and the importance of intestinal heterogeneity in assessing viral responses.