BACKGROUND:Heart failure is a major global health challenge, and regeneration of damaged myocardium remains limited. Mesenchymal stem cells, particularly adipose-derived mesenchymal stem cells, have potential for cardiac regeneration; however, efficient strategies for cardiac differentiation remain unclear. Resveratrol has been reported to promote cardiac differentiation and improve cell survival, but its modulatory effect on 5-azacytidine-induced cardiac differentiation in human adipose-derived mesenchymal stem cells remains unclear. This study aimed to evaluate the effect of resveratrol on 5-azacytidine-induced cardiac differentiation. METHODS:Cells were treated with 5-azacytidine alone or in combination with different concentrations of resveratrol (5,10, and 20 μM). Cell viability was measured using the MTT assay after 72 h, and cardiac gene expression was analyzed using real-time PCR after 21 days. Cells were examined by an inverted microscope. RESULTS:5-azacytidine significantly reduced cell viability, while resveratrol partially alleviated this cytotoxicity. Resveratrol enhanced the expression of cardiac-related genes, including TNNT2, MYOD1, and GATA4. However, co-administration of both agents did not improve the reduced efficacy for some markers. Microscopic examination revealed cellular changes, including perinuclear clear regions that appeared to be consistent with cytoplasmic lipid droplet accumulation in several groups. The Res 20 group showed cell-to-cell junctions morphologically resembling intercalated disc-like structures, suggesting morphological changes associated with a cardiomyocyte-like phenotype. No spontaneous beating was observed in any group during the induction period. CONCLUSION:Resveratrol improved the cardiac induction profile by enhancing the expression of cardiac-related genes. Although it showed a trend toward reducing 5-azacytidine-induced cytotoxicity, this effect was not statistically significant.
Early vertebrate development involves several highly orchestrated morphogenic movements. Two key movements are gastrulation, which organizes germ layer spatial arrangement, and neurulation, which results in the establishment of the neural tube. In this study, we demonstrate that the POZ-ZF transcription factor xZnf131 is required for proper morphogenetic movements during gastrulation and neurulation in Xenopus laevis. xZnf131 is expressed throughout early Xenopus development, with enriched localization in dorsal and anterior embryonic structures, including the neural plate. Morpholino oligonucleotide-mediated depletion of xZnf131 results in defects in axial mesoderm extension as well as neural tube formation. Together, these findings establish xZnf131 as a critical regulator required for the proper morphogenetic movements during early Xenopus development.
Endocardial cushions, derived from the atrioventricular canal (AVC) and outflow tract (OFT), serve as the primordia for the formation of the septum and valves. Improper morphogenesis of the endocardial cushion leads to septal and valvular abnormalities, which contribute to the largest proportion of congenital heart diseases (CHDs). The development and maturation of the endocardial cushion rely on endothelial-to-mesenchymal transition (EndMT), a process by which endocardial cells undergo morphological remodeling and develop into the endocardial cushion mesenchyme. This biological event is strictly governed by regional signals originating from the myocardium, endocardium, and mesenchyme. Various signaling pathways, including TGFβ/BMP, Wnt, Notch, Hippo/YAP, PI3K/AKT, VEGF, and mechanical stress, are involved in the modulation of endocardial cell proliferation, migration, and mesenchymal transition. Transcription factors, such as SMAD, SNAIL, TWIST, TBX2, SOX9, KLF2/4, and NFATc1, exert distinct effects on endocardial EndMT. Maternal environmental exposures can disrupt signaling pathways and transcriptional network during endocardial cushion development, thereby enhancing susceptibility to CHDs. This review summarizes the mechanistic pathways and transcriptional regulation in AVC endocardial cushion development and EndMT, highlighting their association with CHDs.
Human THP-1 monocyte-like cells are a commonly used model for monocyte-derived macrophages (MoDM) in vitro, and significant literature has demonstrated how MoDM phenotype depends on the type, concentration, and duration of differentiation stimuli. However, far less attention has been given to how the choice of differentiation protocols actively reprograms transcriptional signaling networks during the differentiation process. Monocytes begin transcriptional and signaling rewiring immediately upon exposure to differentiation cues, suggesting that these early events condition how macrophages later respond to inflammatory and polarizing stimuli. As a result, differentiation protocols should be considered not as equally neutral preparatory steps, but as biologically active variables that modulate downstream signaling behavior. We compared three differentiation stimuli: PMA, GM-CSF, and M-CSF, to investigate how each agent influenced the temporal activity of key transcription factors. We generated THP-1 cells expressing bioluminescent reporters for CMV, NF-κB and STAT6, enabling longitudinal, quantitative assessment of transcription factor activity throughout differentiation. We found that PMA and GM-CSF enhanced NF-κB activation, whereas M-CSF led to higher STAT6 activity. We also observed that PMA induced early spikes in NF-κB activity and GM-CSF delayed NF-κB activation at later differentiation stages, revealing distinct temporal signaling programs. Differentiation stimuli also induced early-stage downregulation of CMV promoter activity, underscoring global transcriptional remodeling during macrophage maturation. Cytokine assays following secondary polarization demonstrated that CSF-differentiated macrophages exhibited greater responsiveness and dynamic range compared with PMA-differentiated cells. Together, these findings demonstrate that THP-1 differentiation protocols encode stimulus- and time-dependent transcriptional states that directly impact macrophage function. This study establishes temporal transcription factor profiling as a powerful framework for selecting differentiation strategies and experimental timepoints, improving biological relevance and reproducibility in in vitro macrophage models.
Precise regulation of pre-mRNA splicing is essential for normal development, and its disruption represents an important but frequently underrecognized mechanism of human disease. The C-type natriuretic peptide (CNP) receptor NPR2 is a critical regulator of growth plate chondrocyte proliferation and differentiation, and loss-of-function variants in NPR2 cause acromesomelic dysplasia, Maroteaux type (AMDM). Here, we identify a homozygous synonymous NPR2 variant (NM_003995.4:c.2484C > T) in an individual with AMDM and demonstrate its pathogenic mechanism at the RNA level. Although predicted to be silent at the protein level, in silico analysis suggested splice donor gain. Functional analysis using patient-derived leukocyte RNA revealed aberrant splicing leading to partial exon truncation, frameshift, and premature termination of NPR2 which is predicted to trigger nonsense-mediated mRNA decay given its position upstream of multiple downstream exon-exon junctions. Heterozygous family members expressed both normal and aberrant transcripts, whereas the affected individual showed exclusive expression of the aberrant isoform, consistent with a dosage-dependent loss-of-function mechanism. These findings establish aberrant splicing induced by a synonymous variant as a disease-causing mechanism affecting a core developmental signaling pathway. Our study highlights the importance of transcript-level functional analysis in the interpretation of rare variants and underscores the central role of precise RNA processing in skeletal development and human disease.
Polycystic ovary syndrome (PCOS) is a complex heterogeneous endocrine disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. Emerging studies suggest that ovarian fibrosis, as an underrecognized pathological feature, may contribute to ovarian dysfunction and infertility in PCOS phenotypes. Available experimental and limited human studies indicate that hyperandrogenism, insulin resistance, chronic low-grade inflammation, oxidative and endoplasmic reticulum stress collectively promote extracellular matrix accumulation and stromal remodeling predominantly through transforming growth factor-β (TGF-β)-mediated signaling pathways. Additionally, fibrotic remodeling has been linked to other pathways, including the NLRP3 inflammasome, PI3K/Akt, Hippo/YAP, and epithelial-mesenchymal transition. Although standardised diagnostic criteria are currently lacking, new imaging modalities, such as shear-wave elastography, and circulating fibrosis-related biomarkers may offer non-invasive approaches to evaluating ovarian fibrotic alterations. Furthermore, several anti-fibrotic agents, metabolic modulators, phytochemicals, and stem cell-based therapies have been suggested to have potential therapeutic effects in ovarian fibrosis models of PCOS. Overall, ovarian fibrosis represents a developing aspect of PCOS pathophysiology that requires further clinical and translational investigation.
Palmatine, a protoberberine alkaloid with recognized anti-inflammatory and anti-resorptive activities, has recently attracted interest as a potential modulator of bone homeostasis; however, its direct osteo-inductive effects on human adipose-derived mesenchymal stem cells (hADMSCs) remain undefined. Given the accessibility, proliferative efficiency, and clinical relevance of hADMSCs, identifying natural small molecules capable of enhancing their osteogenic differentiation is of considerable therapeutic significance. This study investigated the osteogenic potential of Palmatine using a multi-level in vitro approach. Cytotoxicity assays established a non-toxic working concentration. Osteogenic differentiation was assessed by quantifying alkaline phosphatase (ALP) activity, calcium deposition, and matrix mineralization using Alizarin Red and von Kossa staining. Mechanistic insight was gained by evaluating antioxidant enzyme activity (superoxide dismutase, catalase) and profiling key osteogenic genes via real-time RT-PCR. Low-dose Palmatine (1 μM) enhanced hADMSC viability, whereas higher concentrations displayed dose-dependent cytotoxicity. Functional assays demonstrated that Palmatine significantly increased ALP activity and calcium content at both early (Day 7) and late (Day 14) stages. Transcriptionally, Palmatine promoted early upregulation of Runt-related transcription factor 2, osteocalcin, and osteonectin, followed by robust induction of ALP and collagen type I at later stages, reflecting canonical osteogenic progression. Furthermore, Palmatine augmented antioxidant capacity through early elevation of superoxide dismutase and later activation of catalase, suggesting a redox-associated mechanism that stabilizes osteogenic signaling and supports matrix maturation. Overall, these findings provide the first comprehensive evidence that Palmatine functions as a potent, multi-level osteo-inductive molecule in hADMSCs, highlighting its promise as a natural candidate for redox-modulated bone regenerative strategies.
Precise dorsoventral patterning in Xenopus embryos depends on coordinated transcriptional regulation between organizer and ventral genes. Goosecoid (Gsc) promotes dorsal identity, whereas Ventx1.1, a downstream effector of BMP signaling, specifies ventral fate. Although their antagonistic relationship has been described, the cis-regulatory basis underlying their reciprocal repression has not been clearly defined. Here, we demonstrate that Gsc and Ventx1.1 reciprocally repress each other's transcription through specific cis-regulatory elements within their promoters. Serial promoter deletions and site-directed mutagenesis identified a functional Goosecoid response element (GRE) in the ventx1.1 promoter and Ventx1.1 response elements (VREs) in the gsc promoter. Mutation of these elements markedly reduced reciprocal repression in luciferase assays, and Chromatin immunoprecipitation revealed enrichment at the corresponding promoter regions during gastrulation. In addition, the neuroectoderm-specific transcription factor Foxd4l1.1 also repressed gsc, indicating an additional layer of transcriptional control that refines spatial gene expression. Together, these findings identify a promoter-level reciprocal repression circuit between Gsc and Ventx1.1 and suggest that this circuitry contributes to the refinement of dorsoventral patterning in Xenopus gastrulae.
The prenatal structural organization of the human heel fat pad (HHFP) remains poorly understood, despite its relevance for foot development and related disorders. It is composed of neural, vascular, fibrous, and elastic components arranged into a system of honeycomb-like architecture of microchambers and macrochambers embedded within adipose tissue. This study aimed to characterize the histological and morphometric development of the HHFP, with particular emphasis on compartmentalization and layer-specific maturation, identifying developmental weeks potentially relevant to congenital abnormalities. Embryonic and fetal specimens (8-34 gestational weeks) well preserved anatomical integrity and without histological evidence of malformation or tissue degradation of the feet were included from the Department of Anatomy and Embryology of the Complutense University of Madrid were analyzed. Early stages were characterized by undifferentiated mesenchymal tissue with prominent vascularization and absence of organized adipose structures. Based on structural organization, HHFP development was classified into three stages: Stage A, defined by early organization without a clearly identifiable horizontal fibrous band (HFB); Stage B, representing the progressive formation of the HFB and the onset of compartmentalization into superficial subcutaneous microchamber (SSM) and deep subcutaneous macrochamber (DSM) layers; and Stage C, characterized by a well-defined HFB, established compartmentalization, and progressive adipocyte maturation. Morphometric analysis demonstrated a progressive increase in HHFP thickness, with consistently greater thickness and adipocyte diameter in the DSM compared to the SSM. These findings indicate that HHFP development followed a rapid growth between weeks 14 and 17, with region-specific pattern in which compartmentalization precedes adipocyte maturation and occurs in an asynchronous, layer-dependent manner. This study provides the first structural framework for understanding prenatal HHFP organization, extending beyond classical models of adipose tissue development.
Cartilage and bone that comprise craniofacial structures as well as neurons and glia of the peripheral nervous system are derived from a multipotent population of cranial neural crest cells, that respond to both cell intrinsic and extrinsic cues to differentiate into precise cell states. Both a genetic and epigenetic regulatory network are required for each step in the differentiation process, involving transcription factors, histone modifiers and chromatin remodelers. Here, we examined the direct transcriptional targets of two histone methyltransferases, Prdm3 and Prdm16 in zebrafish neural crest cells at 48 h post fertilization in zebrafish. Using CUT&RUN, we examined both direct DNA binding and nucleosome association. At this stage of development, CUT&RUN fragment size analysis indicated that Prdm3 and Prdm16 are largely associated with nucleosomes. We further analyzed these nucleosome peak sets to identify 6 clusters where differential binding of Prdm3 and Prdm16 and differential enrichment of gene ontology terms for target genes was observed. We validated gene expression in each cluster by in situ hybridization chain reaction (HCR) at 48 hpf demonstrating that prdm3 and prdm16 mutants exhibit corresponding changes in gene expression of the putative gene targets identified. Finally, we performed CUT&RUN-qPCR in prdm3 and prdm16 mutant zebrafish embryos and demonstrated reduced binding at putative target loci. Together these data suggest that Prdm3 and Prdm16 regulate their transcriptional targets primarily by binding nucleosomes around their putative target loci to control downstream gene expression.
Background Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by ectopic bone formation. We investigated in vitro and in vivo the role of developmental endothelial locus-1 (DEL1) on new bone formation and determined the association between DEL1 and spinal progression in AS. Methods DEL1 levels were measured in plasma and facet joint tissues from patients with AS, and in osteoclast-derived medium. Human osteoblast precursor cells were treated with recombinant DEL1 protein and cilengitide trifluoroacetate, an αvβ3 integrin inhibitor, to evaluate their effects on osteoblast differentiation markers. A curdlan-injected SKG mouse model was used to mimic AS pathogenesis. Three weeks after curdlan injection, recombinant DEL1 protein or cilengitide was administered, and the mice's ankle thickness was assessed. The mice were sacrificed after six weeks, and micro-CT and histological analyses were performed. Results DEL1 expression significantly increased during osteoclast differentiation, peaked at the terminal stage, and correlated with disease progression in AS mice. Systemic plasma DEL1 levels were not different between patients with AS and the control group, but were positively correlated with structural damage (mSASSS; R = 0.3433, p = 0.0195). DEL1 pro-osteogenic effects were neutralized by cilengitide, which attenuated DEL1-induced RUNX2 expression and matrix mineralization. Conclusion Our findings establish DEL1 as a key osteoclast-derived coupling factor that drives new bone formation in AS. DEL1 promotes pathological bone formation and osteoblast differentiation by signaling through the integrin αVβ3-RUNX2 axis; targeting this pathway may offer a novel approach to prevent structural damage in patients with AS.
The olfactory sensory system in unique is that the first synaptic contact lies within the central nervous system meaning that the same olfactory sensory neuron whose dendrites are in contact with outside world, has axons extending directly into the central nervous system. Furthermore, neurons of both the peripheral olfactory organ and central olfactory bulb undergo neurogenesis throughout life, although it is unknown whether this is a coordinated process. Here we describe a group of cells in the adult olfactory epithelia that are neuron-like in appearance, yet express the oligodendrocyte precursor marker Olig2:EGFP. These cells extend processes that bypass the olfactory bulbs to arrive at the anterior telencephalic ventricle, the site of precursors that produce interneurons of the olfactory bulbs via the rostral migratory stream. Characterization in sections from intact, wholemount adult brains revealed Sox10 positive cells in patterns consistent with potential migration between the olfactory epithelia and the olfactory bulbs. Based on the expression of Sox10 protein, these cells are assumed to be olfactory ensheathing cells that populate the peripheral and central olfactory system. These cells are a unique type of "hybrid" glia expressing markers associated with astrocytes, oligodendrocytes, and microglia, as well as with progenitor characteristics. Thus, the Olig2:EGFP positive cells link the peripheral olfactory epithelia with the anterior telencephalic vesicle, the source of rostral migratory stream progenitors. These cells provide a potential link to the Sox10+ olfactory ensheathing cells in the adult brain shown here, thus supporting previous findings showing that olfactory ensheathing cells can exert an attractive influence on neuronal migration within the rostral migratory stream.
The tunicate Ciona robusta provides a powerful and simplified model for dissecting the genetic control of developmental and cell biology. With a larval CNS composed of just over 200 neurons and sensory cells, it has also emerged as a model organism for neurobiology and the development of the nervous system. Although CRISPR/ Cas9-mediated mutagenesis is now routinely used in Ciona as an important technique used to interrogate gene function in diverse biological processes, validated single-guide RNAs (sgRNAs) have yet to be validated for several key neural genes. Here, we report the design and experimental validation of 25 novel sgRNAs targeting eight conserved genes encoding conserved proteins involved in neurodevelopment and neural function, including six transcription factors (Cdx, Foxb, Sox1/ 2/3, Dmbx, Engrailed, and Mnx) and two neural effector genes (Tyrosinase and Slc18a3/VAChT). Candidate sgRNAs were selected and tested for mutagenesis efficiency using Illumina-based target site amplicon sequencing. All sgRNAs induced insertions or deletions at their target loci, with most genes yielding at least one sgRNA with mutagenesis efficacy exceeding 30%, with the exception of Dmbx, for which maximal efficacy reached 25%. We further compared measured mutagenesis rates to scores generated by different predictive algorithms, observing a modest but potentially improved correlation with predictions based on a newer algorithm. Based on these results, we recommend considering both scoring algorithms in combination, for improved predictive value for Ciona.
Human periodontal ligament mesenchymal cells (PDLCs) are promising for regenerative therapies but show heterogeneous osteogenic commitment that limits bone regeneration. To attempt to overcome this limitation, we focus on the Hedgehog (Hh) signaling pathway, which is crucial for osteoblastic differentiation as the mechanistic focus of the study and as a strategy to overcome this heterogeneous osteogenic commitment. Resveratrol, a naturally occurring polyphenol, exhibits osteoinductive properties, but its effects on osteogenesis in PDLCs with low osteogenic potential (l-PDLCs) remain unclear. We aimed to investigate whether resveratrol could activate the transcriptional program of Hh signaling in l-PDLCs, as the mechanistic focus of the study and as a strategy to overcome the heterogeneous osteogenic commitment, thereby enhancing mineralization in vitro. Cell viability and osteogenic potential were analyzed under different concentrations and treatment protocols. RNA sequencing, gene expression analysis, transcriptional correlation, and molecular docking were performed in l-PDLCs cultured for 10 days under osteogenic medium (OM) or pretreated for 3 days with resveratrol followed by 10 days of induction (OM/Resv). Pretreatment with 0.1 μM resveratrol exhibited a two-fold increase in mitochondrial metabolism and mineral deposition in vitro. Although multiple osteogenic pathways were modulated, Hh signaling was notably activated, with SHH, DHH, IHH, GLI1 and PTCH1 significantly enriched in OM/Resv compared to OM. Importantly, IHH and SP7 were upregulated and strongly correlated after resveratrol treatment. Resveratrol pretreatment during osteogenic induction upregulated GLI1, PTCH1, and SMO, while downregulating RAB23 (p ≤ 0.01), corroborating RNA-seq results. Molecular docking predicted 23 interactions between resveratrol and RAB23, a negative regulator of Hh signaling, suggesting competition with GDP at the RAB23 active site. Resveratrol enhanced proliferation and osteogenic differentiation in l-PDLCs. Activation of osteogenic markers via the Hh pathway and RAB23 downregulation suggests its role in bone regeneration. These findings provide insights into resveratrol-induced osteogenesis and support its potential as a bioactive modulator of osteogenic pathways in PDLCs for bone regenerative therapies.
Fibrosis, a key pathological process in chronic diseases, involves excessive extracellular matrix deposition that causes organ dysfunction. Due to the limitations of current therapies, identifying new treatment targets is essential. Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that links NAD+ metabolism and epigenetic regulation by consuming nicotinamide (NAM) and S-adenosylmethionine (SAM). This review summarizes the role of NNMT in fibrosis across multiple organs. Evidence shows that NNMT is markedly upregulated in fibrotic tissues, acting as a critical metabolic driver that promotes disease progression through NAD+ depletion, mitochondrial dysfunction, reduced SAM/SAH ratio, fibroblast activation, and feedback loops via pathways like TGF-β1/Smad3. It also exhibits organ-specific mechanisms such as influencing hepatic stellate cells in the liver and cellular senescence in the kidneys. Given its central role in driving fibrogenesis, developing potent and selective NNMT inhibitors has emerged as an urgent therapeutic priority. Current therapeutic strategies targeting NNMT include small-molecule inhibitors, gene-based therapies, and natural compounds. Future work should focus on advancing these inhibitors toward clinical translation and understanding tissue-specific roles of NNMT and its metabolites. Targeting NNMT offers a promising approach for treating fibrosis.
RNA-binding proteins have been established as essential regulators of embryonic development, cellular differentiation and tissue homeostasis. In this review, we highlight the roles of the highly conserved serine/arginine-rich (SR) family member SRSF3 during these processes across a range of organisms. We summarize information regarding the structure and post-translational regulation of SRSF3. Next, we provide an overview of demonstrated roles of SRSF3 in multiple steps of RNA metabolism, including alternative RNA splicing, transcription termination, polyadenylation, stability, transcript nuclear export and miRNA biogenesis. We then discuss the involvement of SRSF3 in various intracellular signaling cascades, including those of the PDGF, EGF, insulin, JAK/STAT, mTOR, retinoic acid, TGF-β and canonical Wnt signaling pathways. Finally, we highlight expression patterns of SRSF3 during embryogenesis and the functions of SRSF3 in the context of mouse germ cell, early embryo, craniofacial, heart and B cell development, as well as in hepatocyte, megakaryocyte and macrophage maturation. We conclude with critical unanswered questions and future directions that should provide significant insight into the roles of SRSF3 in gene expression regulation during development.
Formation of neuronal progenitor cells, neural crest cells and surface ectoderm cells is a complex process and differential gene expression allows for generation of these three cell types during neurulation process. Histone modifiers such as writers and erasers may play an important role in achieving the differential gene expression during neurulation event, similar to how DNMTs and TET enzymes function in germ cell development. We had previously reported that Polycomb group (PcG) protein RING1B which catalysed repressive H2AK119ub1 mark and prevented precocious activation of neuronal genes in undifferentiated cells, but once they were induced to differentiation, RING1B was dislodged from neuronal promoter sites. However, the role of histone H2A deubiquitinases in neuronal differentiation remains to be investigated. Erasers such as histone H2A deubiquitinases Bap1, are critical for normal development since Bap1 KO mice die in embryonic stage. In humans, the cutaneous and uveal melanomas which are ectodermal in origin typically have a mutated BAP1. Recently, it has been shown that Bap1 regulates the epithelial-mesenchymal transition (EMT) genes in developing mice placenta. We differentiated human pluripotent stem cells into the neuronal lineage and depleted BAP1 via doxycycline inducible shRNA. Our results revealed neuronal progenitor markers viz., PAX6, NESTIN and NCAD were reduced at both protein and transcript level upon BAP1 depletion. We observed that BAP1 regulated EMT genes since, cells with depleted BAP1 were deficient in EMT markers such as of SLUG, TWIST1, VIMENTIN and high levels of E-CADHERIN. At the morphological level, this was evident from distorted neuronal rosettes seen post BAP1 knockdown. Bulk RNA seq analysis showed that neuronal progenitor and neural crest specific transcripts were reduced while surface ectoderm transcripts were higher in BAP1 depleted cells. Thus, our results clearly demonstrate that BAP1 is required for human neuronal progenitor cell formation, since it regulates neuronal and EMT genes.
Human brown adipocytes (BAs) derived from stem cells are increasingly recognized as valuable in-vitro models for investigating thermogenesis and metabolic regulation. Despite this promise, existing differentiation strategies show wide variation in cell sources, methodology and validation approaches, limiting reproducibility and translational potential. This systematic review evaluated current protocols for differentiating human pluripotent stem cells (hPSCs) and human adipose-derived stem cells (hADSCs) into brown adipocytes, assessing methodological consistency, differentiation outcomes, and key limitations. A comprehensive search of PubMed and Google Scholar up to May 2024 was conducted following PRISMA 2020 guidelines. Twenty-six studies met inclusion criteria. Protocols using hPSCs revealed broad mechanistic potential but generally yielded immature adipogenic and thermogenic phenotypes, while hADSC-derived models demonstrated more reliable lipid accumulation and UCP1 expression yet remained constrained by procedural variability. Most studies emphasized marker-based validation, with limited incorporation of functional assays. The pronounced methodological heterogeneity across studies precluded quantitative synthesis. Overall, current in-vitro differentiation systems produce only partially functional human brown adipocytes. Establishing standardized validation criteria and reproducible culture frameworks will be critical to enhance comparability, reproducibility, and translational relevance in future research.
In human, an association between bone loss and increased marrow adipose tissue suggests that medullary adipocytes could play a role in osteoporosis by acting on neighboring bone-forming osteoblasts. Supporting this hypothesis, we previously showed, using coculture and conditioned medium models based on human bone marrow stromal cells of commercial origin, that factors secreted by adipocytes induced the transdifferentiation of osteoblasts towards an adipocyte-like phenotype. The aim of this study was to confirm the involvement of medullary adipocyte secretion products in the alteration of osteoblasts in pathophysiological conditions. To this end, two new cellular models were developed from human bone biopsies representing the aging skeleton. In the first model, outgrowth from trabecular bone fragments were used to isolate primary cells that displayed a specific osteoblast phenotype. We confirmed the transdifferentiation of these primary osteoblasts following their incubation with adipocyte conditioned medium as evidenced by the increase in the levels of adipogenic mRNA markers (PPARG, Leptin and HSD11B1, P < 0,001) and the decrease of osteogenic transcript (BGLAP, P < 0.05). The second model was based on primary adipocyte isolation through collagenase treatment followed by ceiling and 2D culture. Oil red O staining confirmed the isolation of fully-differentiated primary adipocytes from bone biopsies. Experiments performed with their conditioned medium validated what had been observed with primary osteoblasts: the effect of the adipocyte secretome on osteoblast fate. Overall, our results supported the relevance of these models to examine paracrine interactions between osteoblasts and adipocytes and confirmed the role played by adipocyte-secreted factors in osteoblast transdifferentiation.
