Patient-specific induced pluripotent stem cells as“disease-in-adish”models for inherited cardiomyopathies and channelopathies–15 years of research

Among inherited cardiac conditions,a special place is kept by cardiomyopathies(CMPs)and channelopathies(CNPs),which pose a substantial healthcare burden due to the complexity of the therapeutic management and cause early mortality.Like other inherited cardiac conditions,genetic CMPs and CNPs exhibit incomplete penetrance and variable expressivity even within carriers of the same pathogenic deoxyribonucleic acid variant,challenging our understanding of the underlying pathogenic mechanisms.Until recently,the lack of accurate physiological preclinical models hindered the investigation of fundamental cellular and molecular mechanisms.The advent of induced pluripotent stem cell(iPSC)technology,along with advances in gene editing,offered unprecedented opportunities to explore hereditary CMPs and CNPs.Hallmark features of iPSCs include the ability to differentiate into unlimited numbers of cells from any of the three germ layers,genetic identity with the subject from whom they were derived,and ease of gene editing,all of which were used to generate“disease-in-a-dish”models of monogenic cardiac conditions.Functionally,iPSC-derived cardiomyocytes that faithfully recapitulate the patient-specific phenotype,allowed the study of disease mechanisms in an individual-/allele-specific manner,as well as the customization of therapeutic regimen.This review provides a synopsis of the most important iPSC-based models of CMPs and CNPs and the potential use for modeling disease mechanisms,personalized therapy and deoxyribonucleic acid variant functional annotation.

Matrix from urine stem cells boosts tissue-specific stem cell mediated functional cartilage reconstruction

Articular cartilage has a limited capacity to self-heal once damaged.Tissue-specific stem cells are a solution for cartilage regeneration;however,ex vivo expansion resulting in cell senescence remains a challenge as a large quantity of high-quality tissue-specific stem cells are needed for cartilage regeneration.Our previous report demonstrated that decellularized extracellular matrix(dECM)deposited by human synovium-derived stem cells(SDSCs),adipose-derived stem cells(ADSCs),urine-derived stem cells(UDSCs),or dermal fibroblasts(DFs)provided an ex vivo solution to rejuvenate human SDSCs in proliferation and chondrogenic potential,particularly for dECM deposited by UDSCs.To make the cell-derived dECM(C-dECM)approach applicable clinically,in this study,we evaluated ex vivo rejuvenation of rabbit infrapatellar fat pad-derived stem cells(IPFSCs),an easily accessible alternative for SDSCs,by the abovementioned C-dECMs,in vivo application for functional cartilage repair in a rabbit osteochondral defect model,and potential cellular and molecular mechanisms underlying this rejuvenation.We found that C-dECM rejuvenation promoted rabbit IPFSCs’cartilage engineering and functional regeneration in both ex vivo and in vivo models,particularly for the dECM deposited by UDSCs,which was further confirmed by proteomics data.RNA-Seq analysis indicated that both mesenchymal-epithelial transition(MET)and inflammation-mediated macrophage activation and polarization are potentially involved in the C-dECM-mediated promotion of IPFSCs’chondrogenic capacity,which needs further investigation.

Decellularized extracellular matrix mediates tissue construction and regeneration

Contributing to organ formation and tissue regeneration,extracellular matrix(ECM)constituents provide tissue with three-dimensional(3D)structural integrity and cellular-function regulation.Containing the crucial traits of the cellular microenvironment,ECM substitutes mediate cell–matrix interactions to prompt stem-cell proliferation and differentiation for 3D organoid construction in vitro or tissue regeneration in vivo.However,these ECMs are often applied generically and have yet to be extensively developed for specific cell types in 3D cultures.Cultured cells also produce rich ECM,particularly stromal cells.Cellular ECM improves 3D culture development in vitro and tissue remodeling during wound healing after implantation into the host as well.Gaining better insight into ECM derived from either tissue or cells that regulate 3D tissue reconstruction or organ regeneration helps us to select,produce,and implant the most suitable ECM and thus promote 3D organoid culture and tissue remodeling for in vivo regeneration.Overall,the decellularization methodologies and tissue/cell-derived ECM as scaffolds or cellular-growth supplements used in cell propagation and differentiation for 3D tissue culture in vitro are discussed.Moreover,current preclinical applications by which ECM components modulate the wound-healing process are reviewed.

Strategies to minimize hypertrophy in cartilage engineering and regeneration

Due to a blood supply shortage,articular cartilage has a limited capacity for selfhealing once damaged.Articular chondrocytes,cartilage progenitor cells,embryonic stem cells,and mesenchymal stem cells are candidate cells for cartilage regeneration.Significant current attention is paid to improving chondrogenic differentiation capacity;unfortunately,the potential chondrogenic hypertrophy of differentiated cells is largely overlooked.Consequently,the engineered tissue is actually a transient cartilage rather than a permanent one.The development of hypertrophic cartilage ends with the onset of endochondral bone formation which has inferior mechanical properties.In this review,current strategies for inhibition of chondrogenic hypertrophy are comprehensively summarized;the impact of cell source options is discussed;and potential mechanisms underlying these strategies are also categorized.This paper aims to provide guidelines for the prevention of hypertrophy in the regeneration of cartilage tissue.This knowledge may also facilitate the retardation of osteophytes in the treatment of osteoarthritis.

Three-dimensional Expansion:In Suspension Culture of SD Rat's Osteoblasts in a Rotating Wall Vessel Bioreactor

Objective To study large-scale expansion of SD （Sprague-Dawley） rat＇s osteoblasts in suspension culture in a rotating wall vessel bioreactor （RWVB）. Methods The bioreactor rotation speeds were adjusted in the range of 0 to 20 rpm, which could provide low shear on the rnicrocarriers around 1 dyn/cm^2. The cells were isolated via sequential digestions of neonatal （less than 3 days old） SD rat calvaria. After the primary culture and several passages, the cells were seeded onto the microcarriers and cultivated in T-flask, spinner flask and RWVB respectively. During the culture period, the cells were counted and observed under the inverted microscope for morphology every 12 h. After 7 days, the cells were evaluated with scanning electron microscope （SEM） for histological examination of the aggregates. Also, the hematoxylin-eosin （HE） staining and alkaline phosphatase （ALP） staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carded out for mineralized nodule formation. Results The results showed that in RWVB, the cells could be expanded by more than ten times and they presented better morphology and vitality and stronger ability to form bones. Conclusions The developed RWVB can provide the culture environment with a relatively low shear force and necessary three-dimensional （3D） interactions among cells and is suitable for osteopath expansion in vitro.

Effect of Aqueous-Alcoholic Extract of Ducrosia Anethifolia Boiss on the Fetal Liver of Diabetic Rats

Objective:Moshgak(Ducrosia anethifolia)is a wild plant with medicinal value.The present study aimed to evaluate the effect of Moshgak on the liver tissue of the diabetic rat fetus.Materials and Methods:In this animal study,the aqueous-alcoholic extract of Moshgak was prepared in the standard method.Forty rats were divided randomly into five groups,including control,sham,and three diabetic groups.The rats were diabetic with intraperitoneal injection of a single dose of streptozotocin(80 mg)and 2 diabetic groups were treated with Moshgak extract(280 and 560 mg/kg/bw)for 19 days.The rats were anesthetized and their blood was taken to measure the blood glucose,insulin,and malondialdehyde.Then,their fetuses were removed.The fetal liver sections were obtained by using the stereological methods.The micrometry of the liver tissue was performed and data were analyzed.Results:The finding showed a statistically significant increase(P<0.01)in the total volume of liver,connective tissue,sinusoid,and hepatocytes in diabetic rats compared to control rats,while these parameters decreased significantly in treated groups with Moshgak.Hepatic cell count hepatic decreased in the treated groups.Furthermore,the changes in blood glucose,malondialdehyde,and insulin in diabetic rats were improved significantly by Moshgak treatment.The dilation of sinusoids,hepatocyte vacuolation,and mild lymphocytosis was observed in all diabetic groups except the treatment group with Moshgak 560 mg/kg/bw.Conclusions:According to obtained results,Moshgak extract was able to compensate partially the changes induced by diabetes in the fetal liver tissue.Therefore,due to the side effects of diabetes during pregnancy,further research on anti-diabetic properties of Moshgak is suggested.

Nidogen:A matrix protein with potential roles in musculoskeletal tissue regeneration

Basement membrane proteins are known to guide cell structures,differentiation,and tissue repair.Although there is a wealth of knowledge on the functions of laminins,per-lecan,and type IV collagen in maintaining tissue homeostasis,not much is known about nidogen.As a key molecule in the basement membrane,nidogen contributes to the formation of a delicate microenvironment that proves necessary for stem cell lineage-specific differentiation.In this review,the expression of nidogen is delineated at both cellular and tissue levels from embryonic to adult stages of development;the effect of nidogens is also summarized in the context of musculoskeletal development and regeneration,including but not limited to adipogenesis,angiogenesis,chondrogenesis,myogenesis,and neurogenesis.Furthermore,potential mechanisms underlying the role of nidogens in stem cell-based tissue regeneration are also discussed.This concise review is expected to facilitate our existing understanding and utilization of nidogen in tissue engineering and regeneration.

Chondrogenic priming of human fetal synovium-derived stem cells in an adult stem cell matrix microenvironment

Cartilage defects are a challenge to treat clinically due to the avascular nature of cartilage.Low immunogenicity and extensive proliferation and multidifferentiation potential make fetal stem cells a promising source for regenerative medicine.In this study,we aimed to determine whether fetal synovium-derived stem cells(FSDSCs)exhibited replicative senescence and whether expansion on decellularized extracellular matrix(dECM)deposited by adult SDSCs(AECM)promoted FSDSCs’chondrogenic potential.FSDSCs from passage 2 and 9 were compared for chondrogenic potential,using Alcian blue staining for sulfated glycosaminoglycans(GAGs),biochemical analysis for DNA and GAG amounts,and real-time PCR for chondrogenic genes including ACAN and COL2A1.Passage 3 FSDSCs were expanded for one passage on plastic flasks(PL),AECM,or dECM deposited by fetal SDSCs(FECM).During expansion,cell proliferation was evaluated using flow cytometry for proliferation index,stem cell surface markers,and resistance to hydrogen peroxide.During chondrogenic induction,expanded FSDSCs were evaluated for tri-lineage differentiation capacity.We found that cell expansion enhanced FSDSCs’chondrogenic potential at least up to passage 9.Expansion on dECMs promoted FSDSCs’proliferative and survival capacity and adipogenic differentiation but not osteogenic capacity.AECM-primed FSDSCs exhibited an enhanced chondrogenic potential.

Cell carrier function of hollow-fiber membrane in rotating wall vessel bioreactor

Large-scale expansion of the osteoblasts of a Sprague-Dawley(SD)rat was studied in a rotating wall hollow-fiber membrane bioreactor(RWHMB)by using hollow-fiber membrane as the carrier.For the sake of contrast,cells were also expanded in a T-flask using a hollow-fiber membrane as carrier and in a rotating wall vessel bioreactor(RWVB)using a microcarrier.During the culture period,the cells were sampled every 12 h,and after 5 days,the cells were harvested and evaluated with scanning electron microscopy(SEM),hematoxylin-eosin(HE)staining and alkaline phosphatase(ALP)staining.Moreover,von-Kossa staining and Alizarin Red S stain-ing were carried out for mineralized nodules formation.The results show that in RWHMB,the cells present better morphology and vitality and secrete much more extracel-lular matrix.It is concluded that the RWHMB combines the advantages of the rotating wall vessel and hollow-fiber membrane bioreactors.The hydrodynamic stimulation within it accelerates the metabolism of the osteoblast and mass transfer,which is propitious to cell differenti-ation and proliferation.

Assessing the response of human primary macrophages to defined fibrous architectures fabricated by melt electrowriting

The dual role of macrophages in the healing process depends on macrophage ability to polarize into phenotypes that can propagate inflammation or exert anti-inflammatory and tissue-remodeling functions.Controlling scaf-fold geometry has been proposed as a strategy to influence macrophage behavior and favor the positive host response to implants.Here,we fabricated Polycaprolactone(PCL)scaffolds by Melt Electrowriting(MEW)to investigate the ability of scaffold architecture to modulate macrophage polarization.Primary human macrophages unpolarized(M0)or polarized into M1,M2a,and M2c phenotypes were cultured on PCL films and MEW scaffolds with pore geometries(square,triangle,and rhombus grid)characterized by different angles.M0,M2a,and M2c macrophages wrapped along the fibers,while M1 macrophages formed clusters with rounded cells.Cell bridges were formed only for angles up to 90◦.No relevant differences were found among PCL films and 3D scaffolds in terms of surface markers.CD206 and CD163 were highly expressed by M2a and M2c macrophages,with M2a macrophages presenting also high levels of CD86.M1 macrophages expressed moderate levels of all markers.The rhombus architecture promoted an increased release by M2a macrophages of IL10,IL13,and sCD163 compared to PCL films.The proangiogenic factor IL18 was also upregulated by the rhombus configuration in M0 and M2a macrophages compared to PCL films.The interesting findings obtained for the rhombus architecture represent a starting point for the design of scaffolds able to modulate macrophage phenotype,prompting investigations addressed to verify their ability to facilitate the healing process in vivo.