Simultaneous binding of bFGF to both FGFR and integrin maintains properties of primed human induced pluripotent stem cells.

Introduction: Basic fibroblast growth factor (bFGF, FGF2) and integrin α6ß1 are important for maintaining the pluripotency of human pluripotent stem cells (hPSCs). Although bFGF-integrin binding contributes to biofunctions in cancer cells, the relationship in hPSCs remains unclear. Methods: To investigate the relationship between bFGF and integrin in human induced pluripotent stem cells (hiPSCs), we generated recombinant human bFGF wild-type and mutant proteins, that do not bind to integrin, FGFR, or both. We then cultured hiPSCs with these recombinant bFGF proteins. To evaluate the abilities of recombinant bFGF proteins in maintaining hPSC properties, pluripotent markers, ERK activity, and focal adhesion structure were analyzed through flow cytometry, immunofluorescence (IF), and immunoblotting (IB). Result: We identified an interaction between bFGF and integrin α6ß1 in vitro and in hiPSCs. The integrin non-binding mutant was incapable of inducing the hPSC properties, such as proliferation, ERK activity, and large focal adhesions at the edges of hiPSC colonies. Signaling induced by bFGF-FGFR binding was essential during the first 24 h after cell seeding for maintaining the properties of hPSCs, followed by a shift towards intracellular signaling via the bFGF-integrin interaction. The mixture of the two bFGF mutants also failed to maintain hPSC properties, indicating that bFGF binds to both FGFR and integrin. Conclusion: Our study demonstrates that the integrin-bFGF-FGFR ternary complex maintains the properties of hPSCs via intracellular signaling, providing insights into the functional crosstalk between bFGF and integrins in hiPSCs.

Gap junction-mediated contraction of myoepithelial cells induces the peristaltic transport of sweat in human eccrine glands.

Eccrine sweat glands play an essential role in regulating body temperature. Sweat is produced in the coiled secretory portion of the gland, which is surrounded by obliquely aligned myoepithelial cells; the sweat is then peristaltically transported to the skin surface. Myoepithelial cells are contractile and have been implicated in sweat transport, but how myoepithelial cells contract and transport sweat remains unexplored. Here, we perform ex vivo live imaging of an isolated human eccrine gland and demonstrate that cholinergic stimulation induces dynamic contractile motion of the coiled secretory duct that is driven by gap junction-mediated contraction of myoepithelial cells. The contraction of the secretory duct occurs segmentally, and it is most prominent in the region surrounded by nerve fibers, followed by distension-contraction sequences of the excretory duct. Overall, our ex vivo live imaging approach provides evidence of the contractile function of myoepithelial cells in peristaltic sweat secretion from human eccrine glands.

Laminin fragments conjugated with perlecan's growth factor-binding domain differentiate human induced pluripotent stem cells into skin-derived precursor cells.

Deriving stem cells to regenerate full-thickness human skin is important for treating skin disorders without invasive surgical procedures. Our previous protocol to differentiate human induced pluripotent stem cells (iPSCs) into skin-derived precursor cells (SKPs) as a source of dermal stem cells employs mouse fibroblasts as feeder cells and is therefore unsuitable for clinical use. Herein, we report a feeder-free method for differentiating iPSCs into SKPs by customising culture substrates. We immunohistochemically screened for laminins expressed in dermal papillae (DP) and explored the conditions for inducing the differentiation of iPSCs into SKPs on recombinant laminin E8 (LM-E8) fragments with or without conjugation to domain I of perlecan (PDI), which binds to growth factors through heparan sulphate chains. Several LM-E8 fragments, including those of LM111, 121, 332, 421, 511, and 521, supported iPSC differentiation into SKPs without PDI conjugation. However, the SKP yield was significantly enhanced on PDI-conjugated LM-E8 fragments. SKPs induced on PDI-conjugated LM111-E8 fragments retained the gene expression patterns characteristic of SKPs, as well as the ability to differentiate into adipocytes, osteocytes, and Schwann cells. Thus, PDI-conjugated LM-E8 fragments are promising agents for inducing iPSC differentiation into SKPs in clinical settings.

Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells.

Polydom is an extracellular matrix protein involved in lymphatic vessel development. Polydom-deficient mice die immediately after birth due to defects in lymphatic vessel remodeling, but the mechanism involved is poorly understood. Here, we report that Polydom directly binds to Tie1, an orphan receptor in the Angiopoietin-Tie axis, and facilitates migration of lymphatic endothelial cells (LECs) in a Tie1-dependent manner. Polydom-induced LEC migration is diminished by PI3K inhibitors but not by an ERK inhibitor, suggesting that the PI3K/Akt signaling pathway is involved in Polydom-induced LEC migration. In line with this possibility, Akt phosphorylation in LECs is enhanced by Polydom although no significant Tie1 phosphorylation is induced by Polydom. LECs also exhibited nuclear exclusion of Foxo1, a signaling event downstream of Akt activation, which was impaired in Polydom-deficient mice. These findings indicate that Polydom is a physiological ligand for Tie1 and participates in lymphatic vessel development through activation of the PI3K/Akt pathway.

Visualization of basement membranes by a nidogen-based fluorescent reporter in mice.

Basement membranes (BMs) are thin, sheet-like extracellular structures that cover the basal side of epithelial and endothelial tissues and provide structural and functional support to adjacent cell layers. The molecular structure of BMs is a fine meshwork that incorporates specialized extracellular matrix proteins. Recently, live visualization of BMs in invertebrates demonstrated that their structure is flexible and dynamically rearranged during cell differentiation and organogenesis. However, the BM dynamics in mammalian tissues remain to be elucidated. We developed a mammalian BM imaging probe based on nidogen-1, a major BM-specific protein. Recombinant human nidogen-1 fused with an enhanced green fluorescent protein (Nid1-EGFP) retains its ability to bind to other BM proteins, such as laminin, type IV collagen, and perlecan, in a solid-phase binding assay. When added to the culture medium of embryoid bodies derived from mouse ES cells, recombinant Nid1-EGFP accumulated in the BM zone of embryoid bodies, and BMs were visualized in vitro. For in vivo BM imaging, a knock-in reporter mouse line expressing human nidogen-1 fused to the red fluorescent protein mCherry (R26-CAG-Nid1-mCherry) was generated. R26-CAG-Nid1-mCherry showed fluorescently labeled BMs in early embryos and adult tissues, such as the epidermis, intestine, and skeletal muscles, whereas BM fluorescence was unclear in several other tissues, such as the lung and heart. In the retina, Nid1-mCherry fluorescence visualized the BMs of vascular endothelium and pericytes. In the developing retina, Nid1-mCherry fluorescence labeled the BM of the major central vessels; however, the BM fluorescence were hardly observed in the peripheral growing tips of the vascular network, despite the presence of endothelial BM. Time-lapse observation of the retinal vascular BM after photobleaching revealed gradual recovery of Nid1-mCherry fluorescence, suggesting the turnover of BM components in developing retinal blood vessels. To the best of our knowledge, this is the first demonstration of in vivo BM imaging using a genetically engineered mammalian model. Although R26-CAG-Nid1-mCherry has some limitations as an in vivo BM imaging model, it has potential applications in the study of BM dynamics during mammalian embryogenesis, tissue regeneration, and pathogenesis.

A heterozygous LAMA5 variant may contribute to slowly progressive, vinculin-enhanced familial FSGS and pulmonary defects.

The LAMA5 gene encodes laminin α5, an indispensable component of glomerular basement membrane and other types of basement membrane. A homozygous pathological variant in LAMA5 is known to cause a systemic developmental syndrome including glomerulopathy. However, the roles of heterozygous LAMA5 gene variants in human renal and systemic diseases have remained unclear. We performed whole-exome sequencing analyses of a family with slowly progressive nephropathy associated with hereditary focal segmental glomerulosclerosis, and we identified what we believe to be a novel probable pathogenic variant of LAMA5, NP_005551.3:p.Val3687Met. In vitro analyses revealed cell type-dependent changes in secretion of variant laminin α5 laminin globular 4-5 (LG4-5) domain. Heterozygous and homozygous knockin mice with a corresponding variant of human LAMA5, p.Val3687Met, developed focal segmental glomerulosclerosis-like pathology with reduced laminin α5 and increased glomerular vinculin levels, which suggested that impaired cell adhesion may underlie this glomerulopathy. We also identified pulmonary defects such as bronchial deformity and alveolar dilation. Reexaminations of the family revealed phenotypes compatible with reduced laminin α5 and increased vinculin levels in affected tissues. Thus, the heterozygous p.Val3687Met variant may cause a new syndromic nephropathy with focal segmental glomerulosclerosis through possibly defective secretion of laminin α5. Enhanced vinculin may be a useful disease marker.

Dynamics, structure and assembly of the basement membrane in developing salivary glands revealed by an exogenous EGFP-tagged nidogen probe.

Most epithelial tissues rapidly become complex during embryonic development while being surrounded by the basement membrane (BM). Thus, the BM shape is thought to change dramatically as the epithelium grows, but the underlying mechanism is not yet clear. Nidogen-1 is ubiquitous in the BM and binds to various other BM components, including laminin and type IV collagen. To elucidate the behavior of the BM during epithelial morphogenesis, we attempted to live-label the developing BM with recombinant human nidogen-1 fused to an enhanced green fluorescent protein (hNid1-EGFP). Submandibular glands of mouse embryos were cultured in glass-bottomed dishes and incubated in media containing hNid1-EGFP. Subsequent confocal microscopy clearly visualized the BMs surrounding the epithelial end buds. On three-dimensional reconstruction from Z-series confocal sections, the epithelial BM was observed as a thin sheet that expanded continuously around the entire epithelial basal surface. Because the explants continued to grow well in the presence of hNid1-EGFP, time-lapse confocal microscopy was performed to follow the dynamics of the BM. We found that the epithelial BM is an adaptive structure that deforms in accordance with the rapid shape changes of the developing epithelium. Furthermore, hNid1-EGFP was found to be incorporated differently into the epithelial BM compared with that reported for fibronectin or type IV collagen, suggesting that individual BM components assemble in different ways to form the BM.

Pretreatment with Perlecan-Conjugated Laminin-E8 Fragment Enhances Maturation of Grafted Dopaminergic Progenitors in Parkinson's Disease Model.

The therapeutic effect of a cell replacement therapy for Parkinson's disease (PD) depends on the proper maturation of grafted dopaminergic (DA) neurons and their functional innervation in the host brain. In the brain, laminin, an extracellular matrix protein, regulates signaling pathways for the survival and development of neurons by interacting with integrins. The heparan sulfate (HS) chain binds mildly to various neurotrophic factors and regulates their intracellular signaling. Perlecan-conjugated laminin 511/521-E8 fragments (p511/p521) were designed to contain an integrin-binding site and HS chains. Here we examined the effect of treating DA progenitors with p511/p521 prior to transplantation in rodent PD models. In vitro and in vivo experiments showed that p511/p521 treatment enhanced the maturation and neurite extension of the grafted DA progenitors by activating RAS-ERK1/2 signaling. This strategy will contribute to an efficient cell replacement therapy for PD in the future.

Establishment and characterization of immortalized sweat gland myoepithelial cells.

Sweat glands play an important role in thermoregulation via sweating, and protect human vitals. The reduction in sweating may increase the incidence of hyperthermia. Myoepithelial cells in sweat glands exhibit stemness characteristics and play a major role in sweat gland homeostasis and sweating processes. Previously, we successfully passaged primary myoepithelial cells in spheroid culture systems; however, they could not be maintained for long under in vitro conditions. No myoepithelial cell line has been established to date. In this study, we transduced two immortalizing genes into primary myoepithelial cells and developed a myoepithelial cell line. When compared with primary sweat gland cells, the immortalized myoepithelial cells (designated "iEM") continued to form spheroids after the 4th passage and expressed α-smooth muscle actin and other proteins that characterize myoepithelial cells. Furthermore, treatment with small compounds targeting the Wnt signaling pathways induced differentiation of iEM cells into luminal cells. Thus, we successfully developed an immortalized myoepithelial cell line having differentiation potential. As animal models are not useful for studying human sweat glands, our cell line will be helpful for studying the mechanisms underlying the pathophysiology of sweating disorders.

Combined administration of laminin-221 and prostacyclin agonist enhances endogenous cardiac repair in an acute infarct rat heart.

Although endogenous cardiac repair by recruitment of stem cells may serve as a therapeutic approach to healing a damaged heart, how to effectively enhance the migration of stem cells to the damaged heart is unclear. Here, we examined whether the combined administration of prostacyclin agonist (ONO1301), a multiple-cytokine inducer, and stem cell niche laminin-221 (LM221), enhances regeneration through endogenous cardiac repair. We administered ONO1301- and LM221-immersed sheets, LM221-immersed sheets, ONO1301-immersed sheets, and PBS-immersed sheets (control) to an acute infarction rat model. Four weeks later, cardiac function, histology, and cytokine expression were analysed. The combined administration of LM221 and ONO1301 upregulated angiogenic and chemotactic factors in the myocardium after 4 weeks and enhanced the accumulation of ILB4 positive cells, SMA positive cells, and platelet-derived growth factor receptor alpha (PDGFRα) and CD90 double-positive cells, leading to the generation of mature microvascular networks. Interstitial fibrosis reduced and functional recovery was prominent in LM221- and ONO1301-administrated hearts as compared with those in ONO1301-administrated or control hearts. LM221 and ONO1301 combination enhanced recruitment of PDGFRα and CD90 double-positive cells, maturation of vessels, and functional recovery in rat acute myocardial infarction hearts, highlighting a new promising acellular approach for the failed heart.

Structural mechanism of laminin recognition by integrin.

Recognition of laminin by integrin receptors is central to the epithelial cell adhesion to basement membrane, but the structural background of this molecular interaction remained elusive. Here, we report the structures of the prototypic laminin receptor α6ß1 integrin alone and in complex with three-chain laminin-511 fragment determined via crystallography and cryo-electron microscopy, respectively. The laminin-integrin interface is made up of several binding sites located on all five subunits, with the laminin γ1 chain C-terminal portion providing focal interaction using two carboxylate anchor points to bridge metal-ion dependent adhesion site of integrin ß1 subunit and Asn189 of integrin α6 subunit. Laminin α5 chain also contributes to the affinity and specificity by making electrostatic interactions with large surface on the ß-propeller domain of α6, part of which comprises an alternatively spliced X1 region. The propeller sheet corresponding to this region shows unusually high mobility, suggesting its unique role in ligand capture.

Mapping the molecular and structural specialization of the skin basement membrane for inter-tissue interactions.

Inter-tissue interaction is fundamental to multicellularity. Although the basement membrane (BM) is located at tissue interfaces, its mode of action in inter-tissue interactions remains poorly understood, mainly because the molecular and structural details of the BM at distinct inter-tissue interfaces remain unclear. By combining quantitative transcriptomics and immunohistochemistry, we systematically identify the cellular origin, molecular identity and tissue distribution of extracellular matrix molecules in mouse hair follicles, and reveal that BM composition and architecture are exquisitely specialized for distinct inter-tissue interactions, including epithelial-fibroblast, epithelial-muscle and epithelial-nerve interactions. The epithelial-fibroblast interface, namely, hair germ-dermal papilla interface, makes asymmetrically organized side-specific heterogeneity in the BM, defined by the newly characterized interface, hook and mesh BMs. One component of these BMs, laminin α5, is required for hair cycle regulation and hair germ-dermal papilla anchoring. Our study highlights the significance of BM heterogeneity in distinct inter-tissue interactions.

Human disease-associated extracellular matrix orthologs ECM3 and QBRICK regulate primary mesenchymal cell migration in sea urchin embryos.

Sea urchin embryos have been one of model organisms to investigate cellular behaviors because of their simple cell composition and transparent body. They also give us an opportunity to investigate molecular functions of human proteins of interest that are conserved in sea urchin. Here we report that human disease-associated extracellular matrix orthologues ECM3 and QBRICK are necessary for mesenchymal cell migration during sea urchin embryogenesis. Immunofluorescence has visualized the colocalization of QBRICK and ECM3 on both apical and basal surface of ectoderm. On the basal surface, QBRICK and ECM3 constitute together a mesh-like fibrillar structure along the blastocoel wall. When the expression of ECM3 was knocked down by antisense-morpholino oligonucleotides, the ECM3-QBRICK fibrillar structure completely disappeared. When QBRICK was knocked down, the ECM3 was still present, but the basally localized fibers became fragmented. The ingression and migration of primary mesenchymal cells were not critically affected, but their migration at later stages was severely affected in both knock-down embryos. As a consequence of impaired primary mesenchymal cell migration, improper spicule formation was observed. These results indicate that ECM3 and QBRICK are components of extracellular matrix, which play important role in primary mesenchymal cell migration, and that sea urchin is a useful experimental animal model to investigate human disease-associated extracellular matrix proteins.

Extracellular laminin regulates hematopoietic potential of pluripotent stem cells through integrin ß1-ILK-ß-catenin-JUN axis.

Recombinant matrices have enabled feeder cell-free maintenance cultures of human pluripotent stem cells (hPSCs), with laminin 511-E8 fragment (LM511-E8) being widely used. However, we herein report that hPSCs maintained on LM511-E8 resist differentiating to multipotent hematopoietic progenitor cells (HPCs), unlike hPSCs maintained on LM421-E8 or LM121-E8. The latter two LM-E8s bound weakly to hPSCs compared with LM511-E8 and activated the canonical Wnt/ß-catenin signaling pathway. Moreover, the extracellular LM-E8-dependent preferential hematopoiesis was associated with a higher expression of integrin ß1 (ITGB1) and downstream integrin-linked protein kinase (ILK), ß-catenin and phosphorylated JUN. Accordingly, the lower coating concentration of LM511-E8 or addition of a Wnt/ß-catenin signaling activator, CHIR99021, facilitated higher HPC yield. In contrast, the inhibition of ILK, Wnt or JNK by inhibitors or mRNA knockdown suppressed the HPC yield. These findings suggest that extracellular laminin scaffolds modulate the hematopoietic differentiation potential of hPSCs by activating the ITGB1-ILK-ß-catenin-JUN axis at the undifferentiated stage. Finally, the combination of low-concentrated LM511-E8 and a revised hPSC-sac method, which adds bFGF, SB431542 and heparin to the conventional method, enabled a higher yield of HPCs and higher rate for definitive hematopoiesis, suggesting a useful protocol for obtaining differentiated hematopoietic cells from hPSCs in general.

Clear Evidence of LAMA5 Gene Biallelic Truncating Variants Causing Infantile Nephrotic Syndrome.

Background: Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid-resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only seven patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome, and one patient with SRNS with biallelic LAMA5 missense variants. Methods: We conducted comprehensive gene screening of Japanese patients with severe proteinuria. With the use of targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays. Results: Biallelic truncating variants in the LAMA5 gene (NM_005560) were detected in three patients from two families. All patients presented with proteinuria within 6 months of age. Patients 1 and 2 were siblings possessing a nonsense variant (c.9232C>T, p.[Arg3078*]) and a splice site variant (c.1282 + 1G>A) that led to exon 9 skipping and a frameshift. Patient 3 had a remarkable irregular contour of the glomerular basement membrane. She was subsequently found to have a nonsense variant (c.8185C>T, p.[Arg2720*]) and the same splice site variant in patients 1 and 2. By in vitro heterotrimer formation assays, both truncating variants produced smaller laminin α5 proteins that nevertheless formed trimers with laminin ß1 and γ1 chains. Patient 4 showed SRNS at the age of 8 years, and carried compound heterozygous missense variants (c.1493C>T, p.[Ala498Val] and c.8399G>A, p.[Arg2800His]). Conclusions: Our patients showed clear evidence of biallelic LAMA5 truncating variants causing infantile nephrotic syndrome. We also discerned the clinical and pathologic characteristics observed in LAMA5-related nephropathy. LAMA5 variant screening should be performed in patients with congenital/infantile nephrotic syndrome.

Pathogenic variants in THSD4, encoding the ADAMTS-like 6 protein, predispose to inherited thoracic aortic aneurysm.

PURPOSE: Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease with often unrecognized inherited forms. We sought to identify novel pathogenic variants associated with autosomal dominant inheritance of TAAD. METHODS: We analyzed exome sequencing data from 35 French TAAD families and performed next-generation sequencing capture panel of genes in 1114 unrelated TAAD patients. Functional effects of pathogenic variants identified were validated in cell, tissue, and mouse models. RESULTS: We identified five functional variants in THSD4 of which two heterozygous variants lead to a premature termination codon. THSD4 encodes ADAMTSL6 (member of the ADAMTS/L superfamily), a microfibril-associated protein that promotes fibrillin-1 matrix assembly. The THSD4 variants studied lead to haploinsufficiency or impaired assembly of fibrillin-1 microfibrils. Thsd4+/- mice showed progressive dilation of the thoracic aorta. Histologic examination of aortic samples from a patient carrying a THSD4 variant and from Thsd4+/- mice, revealed typical medial degeneration and diffuse disruption of extracellular matrix. CONCLUSION: These findings highlight the role of ADAMTSL6 in aortic physiology and TAAD pathogenesis. They will improve TAAD management and help develop new targeted therapies.

Laminin-221 Enhances Therapeutic Effects of Human-Induced Pluripotent Stem Cell-Derived 3-Dimensional Engineered Cardiac Tissue Transplantation in a Rat Ischemic Cardiomyopathy Model.

Background Extracellular matrix, especially laminin-221, may play crucial roles in viability and survival of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) after in vivo transplant. Then, we hypothesized laminin-221 may have an adjuvant effect on therapeutic efficacy by enhancing cell viability and survival after transplantation of 3-dimensional engineered cardiac tissue (ECT) to a rat model of myocardial infarction. Methods and Results In vitro study indicates the impacts of laminin-221 on hiPS-CMs were analyzed on the basis of mechanical function, mitochondrial function, and tolerance to hypoxia. We constructed 3-dimensional ECT containing hiPS-CMs and fibrin gel conjugated with laminin-221. Heart function and in vivo behavior were assessed after engraftment of 3-dimensional ECT (laminin-conjugated ECT, n=10; ECT, n=10; control, n=10) in a rat model of myocardial infarction. In vitro assessment indicated that laminin-221 improves systolic velocity, diastolic velocity, and maximum capacity of oxidative metabolism of hiPS-CMs. Cell viability and lactate dehydrogenase production revealed that laminin-221 improved tolerance to hypoxia. Furthermore, analysis of mRNA expression revealed that antiapoptotic genes were upregulated in the laminin group under hypoxic conditions. Left ventricular ejection fraction of the laminin-conjugated ECT group was significantly better than that of other groups 4 weeks after transplantation. Laminin-conjugated ECT transplantation was associated with significant improvements in expression levels of rat vascular endothelial growth factor. In early assessments, cell survival was also improved in laminin-conjugated ECTs compared with ECT transplantation without laminin-221. Conclusions In vitro laminin-221 enhanced mechanical and metabolic function of hiPS-CMs and improved the therapeutic impact of 3-dimensional ECT in a rat ischemic cardiomyopathy model. These findings suggest that adjuvant laminin-221 may provide a clinical benefit to hiPS-CM constructs.

Cell-Type-Specific Adhesiveness and Proliferation Propensity on Laminin Isoforms Enable Purification of iPSC-Derived Corneal Epithelium.

A treatment for intractable diseases is expected to be the replacement of damaged tissues with products from human induced pluripotent stem cells (hiPSCs). Target cell purification is a critical step for realizing hiPSC-based therapy. Here, we found that hiPSC-derived ocular cell types exhibited unique adhesion specificities and growth characteristics on distinct E8 fragments of laminin isoforms (LNE8s): hiPSC-derived corneal epithelial cells (iCECs) and other non-CECs rapidly adhered preferentially to LN332/411/511E8 and LN211E8, respectively, through differential expression of laminin-binding integrins. Furthermore, LN332E8 promoted epithelial cell proliferation but not that of the other eye-related cells, leading to non-CEC elimination by cell competition. Combining these features with magnetic sorting, highly pure iCEC sheets were fabricated. Thus, we established a simple method for isolating iCECs from various hiPSC-derived cells without using fluorescence-activated cell sorting. This study will facilitate efficient manufacture of iCEC sheets for corneal disease treatment and provide insights into target cell-specific scaffold selection.

A Novel Fluorescent Reporter System Identifies Laminin-511/521 as Potent Regulators of Cardiomyocyte Maturation.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) hold great promise for disease modeling and drug discovery. However, PSC-CMs exhibit immature phenotypes in culture, and the lack of maturity limits their broad applications. While physical and functional analyses are generally used to determine the status of cardiomyocyte maturation, they could be time-consuming and often present challenges in comparing maturation-enhancing strategies. Therefore, there is a demand for a method to assess cardiomyocyte maturation rapidly and reproducibly. In this study, we found that Myomesin-2 (Myom2), encoding M-protein, is upregulated postnatally, and based on this, we targeted TagRFP to the Myom2 locus in mouse embryonic stem cells. Myom2-RFP+ PSC-CMs exhibited more mature phenotypes than RFP- cells in morphology, function and transcriptionally, conductive to sarcomere shortening assays. Using this system, we screened extracellular matrices (ECMs) and identified laminin-511/521 as potent enhancers of cardiomyocyte maturation. Together, we developed and characterized a novel fluorescent reporter system for the assessment of cardiomyocyte maturation and identified potent maturation-enhancing ECMs through this simple and rapid assay. This system is expected to facilitate use of PSC-CMs in a variety of scientific and medical investigations.