Future Regenerative Therapies Research Articles

Single-Cell Transcriptomic Analysis of Salivary Gland Endothelial Cells

Vascular endothelial cells have important tissue-specific functions in fibrosis and regeneration. In the salivary gland, endothelial cells are required for proper development, but their roles within adult glands are largely unknown. To identify ligand–receptor interactions between endothelial cells and other cell types that may be important during fibrosis and regeneration, we used a reversible ductal ligation injury. To induce injury, a clip was applied to the primary ducts for 14 d, and to induce a regenerative response, the clip was subsequently removed for 5 d. To identify endothelial cell-produced factors, we used single-cell RNA sequencing of stromal-enriched cells from adult female submandibular and sublingual salivary glands. Transcriptional profiles of homeostatic salivary gland endothelial cells were compared to endothelial cells of other organs. Salivary gland endothelial cells expressed many unique genes and displayed the highest overlap in gene expression with other fenestrated endothelial cells from the colon, small intestine, and kidney. Comparison of the 14-d ligated, mock-ligated, and 5-d deligated stromal-enriched transcripts and lineage tracing revealed that endothelial cells retain their identity following ligation and recovery from injury. CellChat and NATMI were used to predict changes in ligand–receptor interactions from endothelial cells to other cells in response to ligation and deligation. CellChat and NATMI predicted that after ligation, interactions with fibroblasts, epithelial cells, and glial cells were increased, and following deligation, interactions with pericyte, glia, fibroblasts, and immune cells were increased. Some of the highest-ranked interactions predicted in ligated compared to mock endothelial cells were between glial cells via Col4a2-Cd93 and Jag2-Notch1, as well as epithelial cells via Pecam1-Cd38, while in deligated compared to ligated endothelial cells, the top interactions were between fibroblasts via Ntf3-Ntrk2, glial cells via Hspg2-Itgb1, and pericytes via Jam2-F11r. Understanding salivary gland endothelial cell signaling will inform future endothelial cell-based regenerative therapies.

Investigation of the effectiveness of gelatin hydrolysate in human iPS-RPE cell suspension transplantation

IntroductionThe retinal pigment epithelium (RPE) plays essential roles in maintaining retinal functions as well as choroidal capillaries and can lead to visual disorders if dysfunctional. Transplantation of human-induced pluripotent stem cell-derived RPE (hiPSC-RPE) is a promising therapy for such RPE impaired conditions including age-related macular degeneration. The challenge with cell suspension transplantation is targeted delivery of graft cells and undesired cell reflux. Gelatin hydrolysate, a soluble variant with specific molecular weight distribution, is examined in this study for its potential use in hiPSC-RPE suspension transplantation, particularly in reducing cell reflux and enhancing RPE engraftment. MethodsA retinal bleb model was created using polydimethylsiloxane (PDMS) soft lithography to quantify cellular reflux. We examined the effects of gelatin hydrolysate on the hiPSC-RPE of various aspects of cell behavior and performance such as cell viability, hypoxia reaction, morphology, induction of inflammation and immune responses. ResultsGelatin hydrolysate at 5 % concentration effectively mitigated cell reflux in vitro mimic, improved cell viability, reduced cell aggregation, and had an inhibitory effect on hypoxic reactions due to cell deposition with hiPSC-RPE. Additionally, gelatin hydrolysate did not affect cell adhesion and morphology, and decreased the expression of major histocompatibility complex class II molecules, which suggests reduced immunogenicity of hiPSC-RPE. ConclusionGelatin hydrolysate is considered a valuable and useful candidate for future regenerative therapies in hiPSC-RPE suspension transplantation.

Dental stem cells and their applications in pediatric dentistry

Regenerative medicine has garnered significant attention due to its transformative impact on disease management and the restoration of optimal bodily functions. Stem cells, a cornerstone of regenerative medicine, are particularly renowned for their remarkable capacity to facilitate tissue regeneration and repair, revolutionizing modern healthcare by offering diverse treatment avenues for multiple conditions. Dental pulp stem cells (DPSCs), a subset of postnatal stem cells, are especially noteworthy for their extensive proliferation and ability to differentiate into various specialized cell types. In the realm of pediatric dentistry, there exists a pressing need for advancements in regenerative medicine. Thorough research in this domain has the potential to propel evidence-based pediatric dentistry, ensuring that children receive tailored, high-quality care. Stem cells hold immense promise in pediatric dentistry by offering less invasive and regenerative solutions for a range of childhood dental issues and congenital anomalies. This research, initiated on 04 October 2023, was instigated following an exhaustive review of existing literature utilizing databases like PubMed, Web of Science, and Cochrane. The literature search encompassed a wide array of medical terminologies. Dental stem cells are categorized into four major types: DPSCs, stem cells from human exfoliated deciduous teeth (SHED), stem cells of the apical papilla (SCAP), and periodontal ligament stem cells (PDLSCs). Pediatric dentistry, encompassing endodontics, orthodontics, periodontics, and the treatment of craniofacial defects, stands to benefit significantly from the potential of dental stem cells. The preservation of dental pulp stem cells extracted from deciduous and permanent teeth through tooth banking offers a source for future regenerative therapies. The preservation process involves multiple comprehensive steps. In summary, dental stem cells present a promising avenue within pediatric dentistry, offering multifaceted applications ranging from pulpal regeneration and dentin repair to orthodontic support and the treatment of craniofacial anomalies.

Bone marrow from periacetabular osteotomies as a novel source for human mesenchymal stromal cells

BackgroundBone marrow-derived mesenchymal stromal cells (BM-MSCs) are used in regenerative medicine and related research involving immunomodulatory, anti-inflammatory, anti-fibrotic and regenerative functions. Isolation of BM-MSCs from samples obtained during total hip arthroplasty (THA) is routinely possible. Advanced age and comorbidities of the majority of patients undergoing THA limit their applicability. Our study aimed to evaluate the potential of bone marrow obtained during periacetabular osteotomy (PAO) as a novel source of BM-MSCs from young donors by analyzing cell yield and cell characteristics.MethodsBone samples were obtained from the anterior Os ilium or superior Os pubis during PAO and from the femoral cavity during primary THA. Isolation of bone marrow-derived mononuclear cells (BM-MNCs) was performed by density gradient centrifugation. The samples from PAO and THA patients were compared in terms of BM-MSC yield, colony formation and the proportion of BM-MSCs within the BM-MNC population using flow cytometry analysis. The cells were characterized based on the expression of BM-MSC-specific surface markers. The functionality of the cells was compared by quantifying post-thaw viability, metabolic activity, proliferation capacity, senescence-associated beta galactosidase (SA-β-gal) expression, trilineage differentiation potential and major secretome proteins.ResultsIsolation of BM-MNCs was possible in a reliable and reproducible manner when using bone from PAO containing more than 0.24 g bone marrow. PAO patients were younger than patients of the THA group. Bone obtained during PAO contained less bone marrow and led to a lower BM-MSC number after the first cell culture passage compared to BM-MSCs obtained during THA. BM-MSCs from PAO samples are characterized by a higher proliferation capacity. This results in a higher yield in cell culture passage two, when normalized to the sample weight. BM-MSCs from PAO patients showed increased secretion of TGF-β1, TIMP2, and VEGF upon osteogenic differentiation. BM-MSCs from PAO and THA patients revealed similar results regarding the onset of SA-β-gal expression and trilineage differentiation capacity.ConclusionsWe suggest that bone obtained during PAO is a promising novel source for BM-MSCs from young donors. Limited absolute cell yield due to low sample weight must be considered in early cell culture passages and might be critical for the range of clinical applications possible for BM-MSCs from this source. The higher proliferation capacity and increased growth factor secretion of BM-MSCs from young donors may be beneficial for future regenerative cell therapies, in vitro models, and tissue engineering.

Long-term survival of LGR5 expressing supporting cells after severe ototoxic trauma in the adult mouse cochlea.

The leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a tissue resident stem cell marker, which it is expressed in supporting cells (SCs) in the organ of Corti in the mammalian inner ear. These LGR5+ SCs can be used as an endogenous source of progenitor cells for regeneration of hair cells (HCs) to treat hearing loss and deafness. We have recently reported that LGR5+ SCs survive 1 week after ototoxic trauma. Here, we evaluated Lgr5 expression in the adult cochlea and long-term survival of LGR5+ SCs following severe hearing loss. Lgr5GFP transgenic mice and wild type mice aged postnatal day 30 (P30) and P200 were used. P30 animals were deafened with a single dose of furosemide and kanamycin. Seven and 28 days after deafening, auditory brainstem responses (ABRs) were recorded. Cochleas were harvested to characterize mature HCs and LGR5+ SCs by immunofluorescence microscopy and quantitative reverse transcription PCR (q-RT-PCR). There were no significant age-related changes in Lgr5 expression when comparing normal-hearing (NH) mice aged P200 with P30. Seven and 28 days after ototoxic trauma, there was severe outer HC loss and LGR5 was expressed in the third row of Deiters' cells and in inner pillar cells. Seven days after induction of ototoxic trauma there was an up-regulation of the mRNA expression of Lgr5 compared to the NH condition; 28 days after ototoxic trauma Lgr5 expression was similar to NH levels. The presence of LGR5+ SCs in the adult mouse cochlea, which persists after severe HC loss, suggests potential regenerative capacity of endogenous cochlear progenitor cells in adulthood. To our knowledge, this is the first study showing not only long-term survival of LGR5+ SCs in the normal and ototoxically damaged cochlea, but also increased Lgr5 expression in the adult mouse cochlea after deafening, suggesting long-term availability of potential target cells for future regenerative therapies.

Genetic and Pharmacological YAP Activation Induces Proliferation and Improves Survival in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiomyocyte loss following myocardial infarction cannot be addressed with current clinical therapies. Cell therapy with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a potential approach to replace cardiomyocyte loss. However, engraftment rates in pre-clinical studies have been low, highlighting a need to refine current iPSC-CM technology. In this study, we demonstrated that inducing Yes-associated protein (YAP) by genetic and pharmacological approaches resulted in increased iPSC-CM proliferation and reduced apoptosis in response to oxidative stress. Interestingly, iPSC-CM maturation was differently affected by each strategy, with genetic activation of YAP resulting in a more immature cardiomyocyte-like phenotype not witnessed upon pharmacological YAP activation. Overall, we conclude that YAP activation in iPSC-CMs enhances cell survival and proliferative capacity. Therefore, strategies targeting YAP, or its upstream regulator the Hippo signalling pathway, could potentially be used to improve the efficacy of iPSC-CM technology for use as a future regenerative therapy in myocardial infarction.

Cerebrolysin recovers diaphragmatic function and reduces injury-associated astrogliosis following a cervical spinal cord hemi-section injury in rats

BackgroundSpinal cord injury (SCI) is widely considered the most disastrous medical condition. With no available treatment to date, SCI continues to cause disabilities to the patients and affect their own and their caregivers' quality of life. Cerebrolysin (CBL) is a neuropeptide preparation derived from purified brain proteins with suggested neuroprotective and neurotrophic properties. CBL showed earlier the ability to target multiple pathways that helped in the improvement of the recovery following different groups of neurological diseases and injuries, including ischemic stroke, traumatic brain injuries, and even neurodegenerative diseases. In the current study, the neuroprotective effect of CBL following a SCI is called into question using a rat model of spinal cord cervical hemi-section validated earlier by our lab and others. Using 32 rats divided into four groups randomly, CBL treatment was implemented for either 7 or 21 days duration, following the cervical spinal cord hemi-section.ResultsFollowing the CBL treatment, rats with cervical cord hemi-section showed functional improvement of diaphragmatic muscle as recorded by electromyography (EMG). In addition, the histopathological assessment of the spinal cord showed improved neuronal viability and reduced astrogliosis at the site of the injury compared to the non-treated group. 21-day treatment showed significant improvement when compared to the shorter 7-day regimen.ConclusionOur data suggest that CBL is capable of protecting and regenerating anterior horn motor neurons with functional recovery of diaphragmatic muscle functions in rats, suggesting the potential use of CBL for future regenerative and neuroprotective therapy following SCI.

Orthotopic Ferret Tracheal Transplantation Using a Recellularized Bioengineered Graft Produces Functional Epithelia.

Tracheal grafts may be necessary to bridge long-segment defects after curative resection for airway obstructions. Bioengineered grafts have emerged as an appealing option, given the possibilities of altering the histologic and cellular profile of the conduit. We previously designed a bioreactor capable of luminally decellularizing and recellularizing a ferret trachea with surface airway epithelia (SAE) basal cells (BCs), and we sought to assess the fate of these grafts when transplanted in an orthotopic fashion. As adjuncts to the procedure, we investigated the use of a vascular endothelial growth factor (VEGF)-laden hydrogel and of immunosuppression (IS) in graft revascularization and viability. IS was shown to limit early graft revascularization, but this effect could be counteracted with VEGF supplementation. Submucosal gland (SMG) loss was shown to be inevitable regardless of the revascularization strategy. Lastly, the bioengineered tracheas survived one month after transplant with differentiation of our implanted BCs that then transitioned into a recipient-derived functional epithelium. The work presented in this manuscript has important implications for future cellular and regenerative therapies.

Patterned Arteriole-Scale Vessels Enhance Engraftment, Perfusion, and Vessel Branching Hierarchy of Engineered Human Myocardium for Heart Regeneration.

Heart regeneration after myocardial infarction (MI) using human stem cell-derived cardiomyocytes (CMs) is rapidly accelerating with large animal and human clinical trials. However, vascularization methods to support the engraftment, survival, and development of implanted CMs in the ischemic environment of the infarcted heart remain a key and timely challenge. To this end, we developed a dual remuscularization-revascularization therapy that is evaluated in a rat model of ischemia-reperfusion MI. This study details the differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for engineering cardiac tissue containing patterned engineered vessels 400 μm in diameter. Vascularized engineered human myocardial tissues (vEHMs) are cultured in static conditions or perfused in vitro prior to implantation and evaluated after two weeks. Immunohistochemical staining indicates improved engraftment of hiPSC-CMs in in vitro-perfused vEHMs with greater expression of SMA+ vessels and evidence of inosculation. Three-dimensional vascular reconstructions reveal less tortuous and larger intra-implant vessels, as well as an improved branching hierarchy in in vitro-perfused vEHMs relative to non-perfused controls. Exploratory RNA sequencing of explanted vEHMs supports the hypothesis that co-revascularization impacts hiPSC-CM development in vivo. Our approach provides a strong foundation to enhance vEHM integration, develop hierarchical vascular perfusion, and maximize hiPSC-CM engraftment for future regenerative therapy.

Mapping of Notch signaling in the developing organ of Corti in common marmosets.

The well-regulated development of the sensory epithelium is essential for hearing. This process involves the specification of a pro-sensory epithelium containing common progenitors that differentiate into hair and supporting cells. Notch signaling is one of the most critical pathways during these processes, and its modification is thought to be a feasible approach for treating hearing loss. Despite interspecies differences between rodents and primates or humans, most of our current knowledge regarding cochlear development has been obtained from rodent models. We therefore examined and mapped the expression patterns of Notch signal components in the developing cochlea of the common marmoset (Callithrix jacchus), a small monkey species native to the New World, a primate model animal. In contrast to the preserved expression patterns of the Notch signaling components in the hair cell differentiation between primates and rodents, we unveiled relatively large interspecies differences during the maturation of supporting cells. This improved knowledge of Notch signaling during primate cochlear development will facilitate the development of future regenerative therapies.

Effect of Long-Term Cryopreservation on the Stemness of Stem Cells of Apical Papilla.

Stem cells of apical papilla (SCAPs) are considered a subpopulation of dental stem cells with unique properties. They originate from a developing tissue, the apical papilla of developing teeth, a characteristic that enhances their stemness. Banking of these stem cells can offer a source of dental stem cells for future regenerative therapies. Until now, only the effect of six months' cryopreservation on SCAPs has been studied. In this study, the long-term (19 months) effect of cryopreservation on SCAPs was examined by means of estimation of their differentiation's capacity, flow cytometry immunophenotypical characterization, and molecular characterization of the main transcriptional factors that coincide with pluripotency. As was indicated from our results, 19-month cryopreservation of SCAPs did not affect negatively their stemness; since no significant difference was observed on their typical fibroblast-like morphology, they retained their differentiation capacity, and no discrepancies were found either on immunophenotypical level or molecular level.

STAT3 suppression and β-cell ablation enhance α-to-β reprogramming mediated by Pdx1.

As diabetes results from the absolute or relative deficiency of insulin secretion from pancreatic β cells, possible methods to efficiently generate surrogate β cells have attracted a lot of efforts. To date, insulin-producing cells have been generated from various differentiated cell types in the pancreas, such as acinar cells and α cells, by inducing defined transcription factors, such as PDX1 and MAFA, yet it is still challenging as to how surrogate β cells can be efficiently generated for establishing future regenerative therapies for diabetes. In this study, we demonstrated that the exogenous expression of PDX1 activated STAT3 in α cells in vitro, and STAT3-null PDX1-expressing α cells in vivo resulted in efficient induction of α-to-β reprogramming, accompanied by the emergence of α-cell-derived insulin-producing cells with silenced glucagon expression. Whereas β-cell ablation by alloxan administration significantly increased the number of α-cell-derived insulin-producing cells by PDX1, STAT3 suppression resulted in no further increase in β-cell neogenesis after β-cell ablation. Thus, STAT3 modulation and β-cell ablation nonadditively enhance α-to-β reprogramming induced by PDX1, which may lead to the establishment of cell therapies for curing diabetes.

Comprehensive Genetic Exploration of Fused Teeth by Whole Exome Sequencing

The dental anomaly fused teeth is defined as the union of two or more teeth. Its aetiology is unclear; to date no studies have investigated its genetic background. Therefore, this study, on the basis of a hypothesised genetic component, investigated the genetic background of patients with fused teeth using whole exome sequencing. Fifteen individuals from six families, including members with and without fused teeth, provided saliva samples that were analysed using whole exome sequencing. Patients with other congenital diseases were excluded from this study. Rare variants were extracted from the sequencing data and filtered by family grouping to identify candidate variants. As a result, ERCC6, OBSCN, SLC27A3, and KIF25 were identified as candidate variants. Our sequencing analysis identified four candidate gene variants associated with fused teeth, which now require further investigation. A genetic basis for the anomaly appears likely. This may assist in understanding the aetiology of fused teeth, which in turn supports better oral care and treatment, as well as future regenerative medicine and gene therapy.

Cell Behavioral Dynamics as a Cue in Optimizing Culture Stabilization in the Bioprocessing of Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) are important for future regenerative medicine therapies. However, in the production of PSCs and derivatives, the control of culture-induced fluctuations in the outcome of cell quality remains challenging. A detailed mechanistic understanding of how PSC behaviors are altered in response to biomechanical microenvironments within a culture is necessary for rational bioprocessing optimization. In this review, we discuss recent insights into the role of cell behavioral and mechanical homeostasis in modulating the states and functions of PSCs during culture processes. We delineate promising ways to manipulate the culture variability through regulating cell behaviors using currently developed tools. Furthermore, we anticipate their potential implementation for designing a culture strategy based on the concept of Waddington's epigenetic landscape that may provide a feasible solution for tuning the culture quality and stability in the bioprocessing space.

Novel hydrogel system eliminates subculturing and improves retention of nonsenescent mesenchymal stem cell populations.

Aim: To compare the physiological behavior of mesenchymal stem/stromal cells (MSCs) within an expandable tissue-mimetic 3D system relative to in vitro expansion in a traditional 2D system. Methods: Adipose-derived MSCs (ASCs) were continuously cultured for 6weeks on either 2D culture plastic or in a 3D hydrogel system that eliminated subculturing. ASCs were assessed for senescence, 'stem-like' MSC markers, and ability for their secretome to augment a secondary cell population. Results: The 3D hydrogel system resulted in an enhanced retention of more regenerative, nonsenescent ASC populations that exhibited increased expression of 'stem-like' MSC surface markers. Conclusion: This study introduces a proof-of-concept design for a novel modular 3D system that can improve in vitro expansion ofstem-like cell populations for future regenerative therapies.

Acceleration Voltage and Spot Size of Advanced Bio Material in Nano scale

Hydrogels, films, micro/nanofibers, and particles, which have recently emerged as advanced biomaterials, have great potential for use as cell/drug carriers for localised drug delivery and as biomimetic scaffolds for future regenerative therapies. Biological properties such as biocompatibility, biodegradability, immunogenicity of biomaterials, and current application strategies are discussed. Finally, the final remarks and prospects for such advanced biomaterials are discussed. This article discusses stem cell biology, biomaterials, and technological approaches, as well as the design of biomaterials and devices used in vivo and in vitro. Generating new functional liver substitutes, improving bone repair processes, neurogenesis, groundbreaking models of cardiac fibrosis, and developing novel venous valve prostheses are some of the specific topics covered. This interdisciplinary approach emphasises how various properties of biomaterials and devices play a role in promoting Nano materials to Modern Technology.

Biological characteristics and pulp regeneration potential of stem cells from canine deciduous teeth compared with those of permanent teeth

BackgroundClinical studies have demonstrated that dental pulp stem cells isolated from permanent teeth (PT-DPSCs) are safe and efficacious for complete pulp regeneration in mature pulpectomized permanent teeth with complete apical closure. Moreover, dental pulp stem cells from deciduous teeth (DT-DPSCs) have also been shown to be useful for pulp regenerative cell therapy of injured immature permanent teeth. However, direct comparisons of the pulp regenerative potential of DT-DPSCs and PT-DPSCs from the same individual have not been performed. This study aimed to compare the differences in stem cell properties and pulp regenerative potential of DT-DPSCs and PT-DPSCs of identical origin.MethodsDT-DPSCs and PT-DPSCs were isolated from the same individual dogs at 4 months and 9 months of age, respectively. The expression of cell surface antigen markers, proliferation and migration activities, and gene expression of stem cell markers, angiogenic/neurotrophic factors and senescence markers were compared. The effects of conditioned medium (CM) derived from these cells on cellular proliferation, migration, angiogenesis, neurite outgrowth and immunosuppression were also compared. Autologous transplantation of DT-DPSCs or PT-DPSCs together with G-CSF was performed to treat pulpectomized teeth in individual dogs. The vascularization and reinnervation of the regenerated pulp tissues were qualitatively and quantitatively compared between groups by histomorphometric analyses.ResultsThe rates of positive CXCR4 and G-CSFR expression in DT-DPSCs were significantly higher than those in PT-DPSCs. DT-DPSCs migrated at a higher rate with/without G-CSF and exhibited increased expression of the stem cell markers Oct3/4 and CXCR4 and the angiogenic factor VEGF and decreased expression of the senescence marker p16 than PT-DPSCs. DT-DPSC-derived CM promoted increased cell proliferation, migration with G-CSF, and angiogenesis compared with PT-DPSC-derived CM; however, no difference was observed in neurite outgrowth or immunosuppression. The regenerated pulp tissues in the pulpectomized teeth were quantitatively and qualitatively similar between the DT-DPSCs and PT-DPSCs transplant groups.ConclusionsThese results demonstrated that DT-DPSCs could be a potential clinical alternative to PT-DPSCs for pulp regenerative therapy. DT-DPSCs can be preserved in an individual cell bank and used for potential future pulp regenerative therapy before the supply of an individual’s own sound discarded teeth has been exhausted.

Evaluation of swelling ability of scaffold combination of chitosan and hydroxypatite

Background: Tissue engineering is an important treatment strategy and used in current and future regenerative the-rapies. To achieve the success of tissue engineering, it is necessary to select a scaffold with certain characteristics. The ability to absorb water or swelling in the scaffold material is one of the properties related to architecture and cell activation. Swelling indicates the ability of the scaffold to absorb or retain water from the scaffold. The swelling ability is expected to absorb water from the surrounding tissue and have an impact on the morphology of the scaffold, espe-cially the combination of chitosan and hydroxyapatite on cell growth. Purpose: Knowing the ability of water absorption (swelling) on the scaffold combination of chitosan and hydroxyapatite for the purpose of bone tissue engineering. Method: Literature review with statistical review method that uses inclusion and exclusion criteria with article design used experimental laboratory in vitro and or experimental laboratory in vivo. Results: There are eight articles show-ing increased swelling ability and five articles showing decreased swelling ability. Conclusion: The combination of chitosan and hydroxyapatite biomaterials can increase and decrease the ability of water absorption on the scaffold so that affects the success of tissue engineering.

Recent advances and challenges in organoid-on-a-chip technology

Conventional 2-dimensional cell culture poorly mimics human-relevant models, which is considered a major challenge in biological research. Organoids are a recent breakthrough in 3-dimensional (3D) in vitro tissue engineering that better reflect the physiological, morphological, and functional properties of in vivo organs (e.g., brain, heart, kidney, lung, and liver). Consequently, organoids are extensively used in various impactful biomedical applications including organ development, disease modeling, and clinical drug testing. However, organoid technology still has several limitations, including low reproducibility, vascularization, limited nutrient uptake and distribution (affecting the level of organoid maturation), lack of standardization, and intra-clonal variability. Efforts have been made to overcome these shortcomings of organoid culture. Microfluidic technology has successfully facilitated the establishment of organoid-on-a-chip systems, which effectively improve the structural and physiological features of organoids in a controlled manner. This review discusses the recent advances and developments in organoid-on-a-chip technology. We hope that this study will motivate researchers to explore the possible engagement between microfluidic devices and self-assembled 3D cell cultures to leverage the enhanced quality of organoids, which will have favorable impacts on future tissue regeneration and regenerative therapies.

Single-cell RNA sequencing reveals PDFGRα+ stromal cell subpopulations that promote proacinar cell differentiation in embryonic salivary gland organoids.

Stromal cells can direct the differentiation of epithelial progenitor cells during organ development. Fibroblast growth factor (FGF) signaling is essential for submandibular salivary gland development. Through stromal fibroblast cells, FGF2 can indirectly regulate proacinar cell differentiation in organoids, but the mechanisms are not understood. We performed single-cell RNA-sequencing and identified multiple stromal cell subsets, including Pdgfra+ stromal subsets expressing both Fgf2 and Fgf10. When combined with epithelial progenitor cells in organoids, magnetic-activated cell-sorted PDGFRα+ cells promoted proacinar cell differentiation similarly to total stroma. Gene expression analysis revealed that FGF2 increased the expression of multiple stromal genes, including Bmp2 and Bmp7. Both BMP2 and BMP7 synergized with FGF2, stimulating proacinar cell differentiation but not branching. However, stromal cells grown without FGF2 did not support proacinar organoid differentiation and instead differentiated into myofibroblasts. In organoids, TGFβ1 treatment stimulated myofibroblast differentiation and inhibited the proacinar cell differentiation of epithelial progenitor cells. Conversely, FGF2 reversed the effects of TGFβ1. We also demonstrated that adult salivary stromal cells were FGF2 responsive and could promote proacinar cell differentiation. These FGF2 signaling pathways may have applications in future regenerative therapies.