Laminin mimetic angiogenic and collagen peptide hydrogel for enhance dermal wound healing.

Laminins are essential in basement membrane architecture and critical in re-epithelialization and angiogenesis. These processes and collagen deposition are vital in skin wound healing. The role of angiogenic peptides in accelerating the wound-healing process has been known. The bioactive peptides could be a potential approach due to their similar effects as growth factors and inherent biocompatible and biodegradable nature with lower cost. They can also recognize ligand-receptor interaction and mimic the extracellular matrix. Here, we report novel angiogenic DYVRLAI, CDYVRLAI, angiogenic-collagen PGPIKVAV, and Ac-PGPIKVAV peptides conjugated sodium carboxymethyl cellulose hydrogel, which was designed from laminin. The designed peptide exhibits a better binding with the α3ß1, αvß3, and α5ß1 integrins and CXCR2 receptor, indicating their angiogenic and collagen binding efficiency. The peptides were evaluated to stimulate wound healing in full-thickness excision wounds in normal and diabetic mice (type II). They demonstrated their efficacy in terms of angiogenesis (CD31), re-epithelialization through regeneration of the epidermis (H&E), and collagen deposition (MT). The synthesized peptide hydrogel (DYVRLAI and CDYVRLAI) showed enhanced wound contraction up to 10.1 % and 12.3 % on day 7th compared to standard becaplermin gel (49 %) in a normal wound model. The encouraging results were also observed with the diabetic model, where these peptides showed a significant decrease of 5.20 and 5.17 % in wound size on day 10th compared to the commercial gel (9.27 %). These outcomes signify that the modified angiogenic peptide is a cost effective, novel peptide motif to promote dermal wound healing in both models.

LAMC2 mitigates ER stress by enhancing ER-mitochondria interaction via binding to MYH9 and MYH10.

Highly proliferative and metastatic tumors are constantly exposed to both intrinsic and extrinsic factors that induce adaptation to stressful conditions. Chronic adaptation to endoplasmic reticulum (ER) ER stress is common to many different types of cancers, and poses a major challenge for acquired drug resistance. Here we report that LAMC2, an extracellular matrix protein upregulated in many types of cancers, is localized in the ER of lung, breast, and liver cancer cells. Under tunicamycin-induced ER stress, protein level of LAMC2 is upregulated. Transfection of cancer cells with LAMC2 resulted in the attenuation of ER stress phenotype, accompanied by elevation in mitochondrial membrane potential as well as reduction in reactive oxygen species (ROS) levels and apoptosis. In addition, LAMC2 forms protein complexes with MYH9 and MYH10 to promote mitochondrial aggregation and increased ER-mitochondria interaction at the perinuclear region. Moreover, overexpression of LAMC2 counteracts the effects of ER stress and promotes tumor growth in vivo. Taken together, our results revealed that in complex with MYH9 and MYH10, LAMC2 is essential for promoting ER-mitochondria interaction to alleviate ER stress and allow cancer cells to adapt and proliferate under stressful conditions. This study provides new insights and highlights the promising potential of LAMC2 as a therapeutic target for cancer treatment.

Recombinant Laminin-511 Fragment (iMatrix-511) Coating Supports Maintenance of Human Nucleus Pulposus Progenitor Cells In Vitro.

The angiopoietin-1 receptor (Tie2) marks specific nucleus pulposus (NP) progenitor cells, shows a rapid decline during aging and intervertebral disc degeneration, and has thus sparked interest in its utilization as a regenerative agent against disc degeneration. However, the challenge of maintaining and expanding these progenitor cells in vitro has been a significant hurdle. In this study, we investigated the potential of laminin-511 to sustain Tie2+ NP progenitor cells in vitro. We isolated cells from human NP tissue (n = 5) and cultured them for 6 days on either standard (Non-coat) or iMatrix-511 (laminin-511 product)-coated (Lami-coat) dishes. We assessed these cells for their proliferative capacity, activation of Erk1/2 and Akt pathways, as well as the expression of cell surface markers such as Tie2, GD2, and CD24. To gauge their regenerative potential, we examined their extracellular matrix (ECM) production capacity (intracellular type II collagen (Col2) and proteoglycans (PG)) and their ability to form spherical colonies within methylcellulose hydrogels. Lami-coat significantly enhanced cell proliferation rates and increased Tie2 expression, resulting in a 7.9-fold increase in Tie2-expressing cell yields. Moreover, the overall proportion of cells positive for Tie2 also increased 2.7-fold. Notably, the Col2 positivity rate was significantly higher on laminin-coated plates (Non-coat: 10.24% (±1.7%) versus Lami-coat: 26.2% (±7.5%), p = 0.010), and the ability to form spherical colonies also showed a significant improvement (Non-coat: 40.7 (±8.8)/1000 cells versus Lami-coat: 70.53 (±18.0)/1000 cells, p = 0.016). These findings demonstrate that Lami-coat enhances the potential of NP cells, as indicated by improved colony formation and proliferative characteristics. This highlights the potential of laminin-coating in maintaining the NP progenitor cell phenotype in culture, thereby supporting their translation into prospective clinical cell-transplantation products.

Fibrin hydrogels fortified with FGF-7/10 and laminin-1 peptides promote regeneration of irradiated salivary glands.

Ionizing radiation, commonly used for head and neck cancer treatment, typically damages the salivary glands, resulting in hyposalivation. The development of treatments to restore this lost function is crucial for improving the quality of life for patients suffering from this condition. To address this clinical need, we have developed an innovative hydrogel by chemically conjugating laminin-1 peptides (A99 and YIGSR) and growth factors, FGF-7 and FGF-10, to fibrin hydrogels. Our results demonstrate that FGF-7/10 and laminin-1 peptides fortified fibrin hydrogel [enhanced laminin-1 peptides fibrin hydrogel (Ep-FH)] promotes salivary gland regeneration and functionality by improving epithelial tissue organization, establishing a healthy network of blood vessels and nerves, while reducing fibrosis in a head and neck irradiated mouse model. These results indicate that fibrin hydrogel-based implantable scaffolds containing pro-regenerative signals promote sustained secretory function of irradiated salivary glands, offering a potential alternative treatment for hyposalivation in head and neck cancer patients undergoing radiation treatment. These unique findings emphasize the potential of fibrin hydrogel-based implantable scaffolds enriched with pro-regenerative signals in sustaining the secretory function of irradiated salivary glands and offer a promising alternative treatment for addressing hyposalivation in head and neck cancer patients undergoing radiation therapy. STATEMENT OF SIGNIFICANCE: Radiation therapies used to treat head and neck cancers often result in damaged salivary gland, leading to severe dryness of the oral cavity. In this study, we engineered FGF-7 and FGF-10 and immobilized them into L1p-FH. The resulting hydrogel, Ep-FH, restored irradiated salivary gland functionality by enhancing epithelial tissue organization, promoting the development of a healthy network of blood vessels and nerves as well as reduction of fibrosis.

ECM proteins and cationic polymers coating promote dedifferentiation of patient-derived mature adipocytes to stem cells.

Reprogramming of mature adipocytes is an attractive research area due to the plasticity of these cells. Mature adipocytes can be reprogrammed in vitro, transforming them into dedifferentiated fat cells (DFATs), which are considered a new type of stem cell, and thereby have a high potential for use in tissue engineering and regenerative medicine. However, there are still no reports or findings on in vitro controlling the dedifferentiation. Although ceiling culture performed in related studies is a relatively simple method, its yield is low and does not allow manipulation of mature adipocytes to increase or decrease the dedifferentiation. In this study, to understand the role of physicochemical surface effects on the dedifferentiation of patient-derived mature adipocytes, the surfaces of cell culture flasks were coated with extracellular matrix, basement membrane proteins, and cationic/anionic polymers. Extracellular matrix such as fibronectin and collagen type I, and basement membrane proteins such as collagen type IV and laminin strongly promoted dedifferentiation of mature adipocytes, with laminin showing the highest effect with a DFAT ratio of 2.98 (±0.84). Interestingly, cationic polymers also showed a high dedifferentiation effect, but anionic polymers did not, and poly(diallyl dimethylammonium chloride) showed the highest DFAT ratio of 2.27 (±2.8) among the cationic polymers. Protein assay results revealed that serum proteins were strongly adsorbed on the surfaces of the cationic polymer coating, including inducing high mature adipocyte adhesion. This study demonstrates for the first time the possibility of regulating the transformation of mature adipocytes to DFAT stem cells by controlling the physicochemical properties of the surface of conventional cell culture flasks.

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.

Xeno-free culture and proliferation of hPSCs on 2D biomaterials.

Human pluripotent stem cells (human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs)) have unlimited proliferative potential, whereas adult stem cells such as bone marrow-derived stem cells and adipose-derived stem cells have problems with aging. When hPSCs are intended to be cultured on feeder-free or xeno-free conditions without utilizing mouse embryonic fibroblasts or human fibroblasts, they cannot be cultured on conventional tissue culture polystyrene dishes, as adult stem cells can be cultured but should be cultivated on material surfaces grafted or coated with (a) natural or recombinant extracellular matrix (ECM) proteins, (b) ECM protein-derived peptides and specific synthetic polymer surfaces in xeno-free and/or chemically defined conditions. This review describes current developing cell culture biomaterials for the proliferation of hPSCs while maintaining the pluripotency and differentiation potential of the cells into 3 germ layers. Biomaterials for the cultivation of hPSCs without utilizing a feeder layer are essential to decrease the risk of xenogenic molecules, which contributes to the potential clinical usage of hPSCs. ECM proteins such as human recombinant vitronectin, laminin-511 and laminin-521 have been utilized instead of Matrigel for the feeder-free cultivation of hPSCs. The following biomaterials are also discussed for hPSC cultivation: (a) decellularized ECM, (b) peptide-grafted biomaterials derived from ECM proteins, (c) recombinant E-cadherin-coated surface, (d) polysaccharide-immobilized surface, (e) synthetic polymer surfaces with and without bioactive sites, (f) thermoresponsive polymer surfaces with and without bioactive sites, and (g) synthetic microfibrous scaffolds.

Cryopreservation Alters Tissue Structure and Improves Differentiation of Engineered Skeletal Muscle.

Tissue-engineered skeletal muscle can play an important role in regenerative medicine, disease modeling, drug testing, as well as the actuation of biohybrid machines. As the applications of engineered muscle tissues expand, there exists a growing need to cryopreserve and store these tissues without impairing function. In a previous study, we developed a cryopreservation protocol in which engineered skeletal muscle tissues are frozen before myogenic differentiation. In that study, we found that this cryopreservation process led to a three-fold increase in the force generation of the differentiated muscle. Here, we perform further testing to determine the mechanisms by which cryopreservation enhances engineered skeletal muscle function. We found that cryopreservation alters the microstructure of the tissue by increasing pore size and decreasing elastic modulus of the extracellular matrix (ECM), which leads to increased expression of genes related to cell migration, cell-matrix adhesion, ECM secretion, and protease activity. Specifically, cryopreservation leads to the upregulation of many ECM proteins, including laminin, fibronectin, and several types of collagens, as well as integrins and matrix metalloproteinases. These changes to ECM structure and composition were associated with enhanced myogenic differentiation, as evidenced by the upregulation of late-stage myogenic markers and increased force generation. These results highlight the need to understand the effects of cryopreservation on the ECM of other tissues as we strive to advance tissue and organ cryopreservation protocols for regenerative medicine.

LAMC2 regulates proliferation, migration, and invasion mediated by the Pl3K/AKT/mTOR pathway in oral.

Background: Oral squamous cell carcinoma (OSCC) is a common malignant tumor. Recently, Laminin Gamma 2 (LAMC2) has been shown to be abnormally expressed in OSCC; however, how LAMC2 signaling contributes to the occurrence and development of OSCC and the role of autophagy in OSCC has not been fully explored. This study aimed to analyze the role and mechanism of LAMC2 signaling in OSCC and the involvement of autophagy in OSCC. Methods: To explore the mechanism by which LAMC2 is highly expressed in OSCC, we used small interfering RNA (siRNA) to knock down LAMC2 to further observe the changes in the signaling pathway. Furthermore, we used cell proliferation assays, Transwell invasion assays, and wound-healing assays to observe the changes in OSCC proliferation, invasion, and metastasis. RFP-LC3 was used to detect the level of autophagy intensity. A cell line-derived xenograft (CDX) model was used to detect the effect of LAMC2 on tumor growth in vivo. Results: This study found that the level of autophagy was correlated with the biological behavior of OSCC. The downregulation of LAMC2 activated autophagy and inhibited OSCC proliferation, invasion, and metastasis via inhibiting the PI3K/AKT/mTOR pathway. Moreover, autophagy has a dual effect on OSCC, and the synergistic downregulation of LAMC2 and autophagy can inhibit OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. Conclusions: LAMC2 interacts with autophagy to regulate OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. LAMC2 down-regulation can synergistically modulate autophagy to inhibit OSCC migration, invasion, and proliferation.

Evaluation of the effects of sinapic acid and ellagic acid on sciatic nerve in experimental diabetic rats by immunohistochemical and stereological methods.

In our study, we aimed to examine the effects of sinapic acid and ellagic acid on neuropathy caused by diabetes in peripheral nerves. Fifty-six adult Wistar Albino rats Control, Diabetes, Diabetes+Sinapic Acid, Diabetes+Ellagic Acid, Diabetes+Sinapic Acid+Ellagic Acid, Sinapic Acid, Ellagic Acid and as Sinapic Acid+Ellagic Acid, they were randomly divided into eight groups(n:7). A single dose of 50 mg/kg streptozotocin(STZ) was administered intraperitoneally to the groups to be diagnosed with diabetes. Diabetes was accepted as blood glucose value of 250 mg/dL and above. Streptozotocin was given to the diabetes groups, 20 mg/kg/day intragastric Sinapic acid to the Sinapic acid groups, 50 mg/kg/day intragastric Ellagic acid to the Ellagic acid groups for 28 days. At the end of the experiment, 0.5 cm of the right sciatic nerve was removed. It was fixed in 10% formaldehyde. After histological follow-up, it was embedded in paraffin, 5 µm thick sections were taken. Immunohistochemical staining with Fibrinogen alpha, Laminin ß-1 and Collagen IV antibodies and stereological evaluation was performed by Physical Dissector Combination method. Collagen IV was used in control, diabetes and treatment groups showed similar immunostaining. Fibrinogen alpha was observed to be increased in the vessel wall in the diabetes group, while the uptake was minimal in the control and treatment groups. While Laminin ß-1 was increased in the diabetes group compared to the control group, immunostaining was observed in the treatment groups similar to the control group. It was observed that the total nerve area diabetes group decreased significantly compared to the control group, and the treatment groups, except for D+EA group were similar to the control group, but there was no statistically significant difference. The axon numbers in the diabetes group decreased significantly compared to the control group, and the treatment groups were similar to the control group, and there was no statistically significant difference (P > 0.05). It was determined that Sinapic Acid and Ellagic acid had positive effects on the nervous tissue in diabetic neuropathy.

A Novel Three-Dimensional Follicle Culture System Decreases Oxidative Stress and Promotes the Prolonged Culture of Human Granulosa Cells.

Tissue engineering advancements have made it possible to modify biomaterials to reconstruct a similar three-dimensional structure of the extracellular matrix (ECM) for follicle development and to supply the required biological signals. We postulated that an artificial polysaccharide hydrogel modified with an ECM mimetic peptide may produce efficient irritation signals by binding to specific integrins providing a suitable environment for follicular development and influencing the behavior of human granulosa cells (hGCs). Laminin, an important component of the extracellular matrix, can modulate hGCs and oocyte growth. Specifically, follicles of mice were randomly divided into two-dimensional (2D) and three-dimensional (3D) culture systems established by a hydrogel modified with RGD or laminin mimetic peptides (IKVAV and YIGSR) and RGD (IYR). Our results showed that 3D cultured systems significantly improved follicle survival, growth, and viability. IYR peptides enhanced the oocyte meiosis competence. Additionally, we explored the effect of 3D culture on hGCs, which improved hGCs viability, increased the proportion of S- and G2/M-phase cells, and inhibited cell apoptosis of hGCs. On days 1 and 2, the secretion of progesterone was reduced in 3D-cultured hGCs. Notably, 3D-cultured hGCs exhibited delayed senescence, decreased oxidative stress, and elevated mitochondrial membrane potential. Moreover, the expression levels of cumulus expansion-related genes (COX2, HAS2, and PTX3) and integrin α6ß1 were upregulated in 3D-cultured hGCs. In conclusion, a 3D culture utilizing hydrogels modified with Laminin-mimetic peptides can provide a durable physical environment suitable for follicular development. The laminin-mimetic peptides may regulate the biological activity of hGCs by attaching to the integrin α6ß1.

Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions.

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFß) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue-laminin-111-switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFß signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFß inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFß inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro.

Systematic Comparison of Commercial Hydrogels Revealed That a Synergy of Laminin and Strain-Stiffening Promotes Directed Migration of Neural Cells.

Hydrogels have shown potential in replacing damaged nerve tissue, but the ideal hydrogel is yet to be found. In this study, various commercially available hydrogels were compared. Schwann cells, fibroblasts, and dorsal root ganglia neurons were seeded on the hydrogels, and their morphology, viability, proliferation, and migration were examined. Additionally, detailed analyses of the gels' rheological properties and topography were conducted. Our results demonstrate vast differences on cell elongation and directed migration on the hydrogels. Laminin was identified as the driver behind cell elongation and in combination with a porous, fibrous, and strain-stiffening matrix structure responsible for oriented cell motility. This study improves our understanding of cell-matrix interactions and thereby facilitates tailored fabrication of hydrogels in the future.

Extracorporeal Shock Wave Therapy Improves Nontraumatic Knee Contracture in a Rat Model.

BACKGROUND: Joint contractures occur frequently after trauma or immobilization, but few reliable treatments are available. Extracorporeal shock wave therapy (ESWT) is often used for various musculoskeletal conditions, but whether it is effective for treating joint contractures and the mechanisms through which it might work for that condition remain unclear. QUESTIONS/PURPOSES: Using a rat model, we asked, does ESWT (1) inhibit the progression of knee contracture, (2) ameliorate histopathologic joint changes, and (3) improve serum and myofascial fibrosis-related factors? We also asked, (4) what is the possible mechanism by which ESWT inhibits knee contracture? METHODS: Thirty-two male Sprague-Dawley rats (12 weeks old and weighing 300 to 400 g) were randomly separated into two groups: control group (eight rats) and noncontrol group (24) in the first week. Rats in the control group were kept free in cages for 4 weeks, and the right lower limbs of the rats in the noncontrol group were immobilized in plaster for 4 weeks. ROM was then measured for each rat with or without 4 weeks of immobilization. After ROM measurement, rats in the noncontrol group were randomly separated into three groups: immobilization group (eight rats), remobilization group (eight rats), and remobilization with ESWT group (eight rats) at Week 4. Knee contracture was induced in rats by fixing the right knee with a plaster cast as in a previous study. The plaster cast was removed after 4 weeks; knee contracture was established when passive ROM was decreased and dysfunction such as abnormal gait occurred. Subsequently, rats with a remobilized joint contracture were treated with or without ESWT for 15 days (on Days 5, 10, and 15). The therapeutic effect was examined using ROM, joint diameter (as an indication of swelling), histopathologic changes, and the levels of fibrosis-related extracellular matrix component factors (hyaluronic acid, serum procollagen peptide, and laminin). The effect of ESWT on fibrosis protein was also evaluated using immunohistochemistry, quantitative polymerase chain reaction (qPCR), and Western blot. The expressions of factors in the TGF-ß/SMADs pathway were also determined using Western blot and qPCR. RESULTS: ESWT mitigated immobilization-induced knee contracture in rats by improving ROM (immobilization versus remobilization with ESWT: 53° ± 8° versus 32° ± 8° [95% confidence interval 13° to 30°]; p < 0.001) and joint swelling (immobilization versus remobilization with ESWT: 8 ± 0.8 cm versus 6 ± 0.3 cm [95% CI 0.4 to 2.2 cm]; p = 0.01). Histopathologic features of remission were alleviated after ESWT (immobilization versus remobilization with ESWT: thickness of the knee space: 0.2 ± 0.03 mm versus 0.6 ± 0.01 mm [95% CI -0.49 to -0.33 mm]; p < 0.001. On Masson staining, the positive expression area, which indicates collagen fiber deposition, was 24% ± 5% versus 9% ± 2% ([95% CI 10% to 21%]; p < 0.001). ESWT improved the serum fibrosis factors of hyaluronic acid, procollagen peptide, and laminin (immobilization versus remobilization with ESWT: hyaluronic acid: 412 ± 32 versus 326 ±15 ng/mL [95% CI 29 to 144 ng/mL]; p = 0.003; serum procollagen peptide: 19 ± 1 versus 12 ±1 ng/mL [95% CI 3 to 11 ng/mL]; p < 0.001; laminin: 624 ± 78 versus 468 ±9 ng/mL [95% CI 81 to 231 ng/mL]; p = 0.006) and myofascial factors of α-SMA and Type I collagen associated with immobilization-induced contractures. CONCLUSION: The findings suggest that ESWT improved joint contracture by inhibiting the TGF-ß1/SMADs signaling pathway in rats. CLINICAL RELEVANCE: This work suggests ESWT may be worth exploring in preliminary research in humans to determine whether it may be a treatment option for patients with nontraumatic knee contractures. If the mechanism of ESWT can be confirmed in humans, ESWT might be a therapy for diseases involved in the TGF-ß1/SMADs signaling pathway, such as hypertroic scarring and scleroderma.

Indirect co-culture of islet cells in 3D biocompatible collagen/laminin scaffold with angiomiRs transfected mesenchymal stem cells.

Diabetes is an autoimmune disease in which the pancreatic islets produce insufficient insulin. One of the treatment strategies is islet isolation, which may damage these cells as they lack vasculature. Biocompatible scaffolds are one of the efficient techniques for dealing with this issue. The current study is aimed to determine the effect of transfected BM-MSCS with angiomiR-126 and -210 on the survival and functionality of islets loaded into a 3D scaffold via laminin (LMN). AngiomiRs/Poly Ethylenimine polyplexes were transfected into bone marrow-mesenchymal stem cells (BM-MSCs), followed by 3-day indirect co-culturing with islets laden in collagen (Col)-based hydrogel scaffolds containing LMN. Islet proliferation and viability were significantly increased in LMN-containing scaffolds, particularly in the miRNA-126 treated group. Insulin gene expression was superior in Col scaffolds, especially, in the BM-MSCs/miRNA-126 treated group. VEGF was upregulated in the LMN-containing scaffolds in both miRNA-treated groups, specifically in the miRNA-210, leading to VEGF secretion. MiRNAs' target genes showed no downregulation in LMN-free scaffolds; while a drastic downregulation was seen in the LMN-containing scaffolds. The highest insulin secretion was recorded in the Oxidized dextran (Odex)/ColLMN+ group with miRNA-126. LMN-containing biocompatible scaffolds, once combined with angiomiRs and their downstream effectors, promote islets survival and restore function, leading to enhanced angiogenesis and glycemic status.

In vitro complete differentiation of human spermatogonial stem cells to morphologic spermatozoa using a hybrid hydrogel of agarose and laminin.

Spermatogenesis refers to the differentiation of the spermatogonial stem cells (SSCs) located in the base seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix (ECM), paracrine factors, and testicular somatic cells which play a supporting role for SSCs. Thus, the present study evaluated the potential of co-culturing Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin, the main components of the ECM. Following the three-week conventional culture of human testicular cells, the cells were cultured in agarose hydrogel or agarose/laminin one (hybrid) for 74 days. Then, immunocytochemistry, real-time PCR, electron microscopy, and morphological staining methods were applied to analyze the presence of SSCs, as well as the other cells of the different stages of spermatogenesis. Based on the results, the colonies with positive spermatogenesis markers were observed in both culture systems. The existence of the cells of all three phases of spermatogenesis (spermatogonia, meiosis, and spermiogenesis) was confirmed in the two groups, while morphological spermatozoa were detected only in the hybrid hydrogel group. Finally, a biologically improved 3D matrix can support all the physiological activities of SSCs such as survival, proliferation, and differentiation.

Extracellular matrix inclusion in immunoisolating alginate-based microcapsules promotes longevity, reduces fibrosis, and supports function of islet allografts in vivo.

Immunoisolation of pancreatic-islets in alginate-microcapsules is applied to treat diabetes. However, long-term islet function is limited, which might be due to damaged and lack of contact with pancreatic extracellular matrix (ECM) components. Herein we investigated the impact of collagen IV combined with laminin sequences, either RGD, LRE, or PDSGR, on graft-survival of microencapsulated bioluminescent islets in vivo. Collagen IV with RGD had the most pronounced effect. It enhanced after 8-week implantation in immune-incompetent mice the bioluminescence of allogeneic islets by 3.2-fold, oxygen consumption rate by 14.3-fold and glucose-induced insulin release by 9.6-fold. Transcriptomics demonstrated that ECM enhanced canonical pathways involving insulin-secretion and that it suppressed pathways related to inflammation and hypoxic stress. Also, 5.8-fold fewer capsules were affected by fibrosis. In a subsequent longevity study in immune-competent mice, microencapsulated allografts containing collagen IV and RGD had a 2.4-fold higher functionality in the first week after implantation and remained at least 2.1-fold higher during the study. Islets in microcapsules containing collagen IV and RGD survived 211 ± 24.1 days while controls survived 125 ± 19.7 days. Our findings provide in vivo evidence for the efficacy of supplementing immunoisolating devices with specific ECM components to enhance functionality and longevity of islet-grafts in vivo. STATEMENT OF SIGNIFICANCE: Limitations in duration of survival of immunoisolated pancreatic islet grafts is a major obstacle for application of the technology to treat diabetes. Accumulating evidence supports that incorporation of extracellular matrix (ECM) molecules in the capsules enhances longevity of pancreatic islets. After selection of the most efficacious laminin sequence in vitro, we show in vivo that inclusion of collagen IV and RGD in alginate-based microcapsules enhances survival, insulin secretion function, and mitochondrial function. It also suppresses fibrosis by lowering proinflammatory cytokines secretion. Moreover, transcriptomic analysis shows that ECM-inclusion promotes insulin-secretion related pathways and attenuates inflammation and hypoxic stress related pathways in islets. We show that inclusion of ECM in immunoisolating devices is a promising strategy to promote long-term survival of islet-grafts.

Epithelial folding of alveolar cells derived from human induced pluripotent stem cells on artificial basement membrane.

Epithelial folding depends on mechanical properties of both epithelial cells and underlying basement membrane (BM). While folding is essential for tissue morphogenesis and functions, it is difficult to recapitulate features of a growing epithelial monolayer for in vitro modeling due to lack of in vivo like BM. Herein, we report a method to overcome this difficulty by culturing on an artificial basement membrane (ABM) the primordial lung progenitors (PLPs) from human induced pluripotent stem cells (hiPSCs). The ABM was achieved by self-assembling collagen IV and laminin, the two principal natural BM proteins, in the pores of a monolayer of crosslinked gelatin nanofibers deposited on a honeycomb micro-frame. The hiPSC-PLPs were seeded on the ABM for alveolar differentiation under submerged and air-liquid interface culture conditions. As results, the forces generated by the growing epithelial monolayer led to a geometry-dependent folding. Analysis of strain distribution in a clamped membrane provided instrumental insights into some of the observed phenomena. Moreover, the forces generated by the growing epithelial layer led to a high-level expression of surfactant protein C and a high percentage of aquaporin 5 positive cells compared with the results obtained with a nanofiber-covered bulk substrate. Thus, this work demonstrated the importance of recapitulating natural BM for advanced epithelial modeling. STATEMENT OF SIGNIFICANCE: The effort to develop in vitro epithelial models has not been entirely successful to date, due to lack of in vivo like basement membrane (BM). This lack has been overcome by using a microfabricated dense thin and pliable sheet like structure made of natural BM proteins. With such an artificial BM, alveolar epithelial deformation and folding could be studied and date could be correlated to numerical analyses of a plate theory. This method is simple and effective, enabling further developments in epithelial tissue modeling.

Comparison of human recombinant protein coatings and fibroblast-ECM to Matrigel for induced pluripotent stem cell culture and renal podocyte differentiation.

Human induced pluripotent stem cells (hiPSCs) offer great opportunities within the 3R framework. In the field of toxicology, they may contribute greatly to the reduction and eventually replacement of animal models. However, culturing hiPSCs as well as differentiation of hiPSCs into target cells that are used for toxicity testing depend on the presence of extracellular matrix (ECM) coating the growth surface. The most widely used ECM is MatrigelR, an animal product that is derived from mouse sarcoma. Drawbacks of Matrigel are widely recognized and include batch-to batch variations, use of animal rather than human material, and ethical concerns about its production. While alternative coatings exist, higher cost and limited characterizations may hinder their broader uptake by the scientific community. Here, we report an extensive comparison of three commercially available human ECM coatings, vitronectin, laminin-511, and laminin-521, to Matrigel in three different hiPSC lines in long-term culture (≥ 9 passages). Characterization included expression of pluripotent markers in a genome-wide transcriptomics study (TempO-Seq), capacity to differentiate into embryoid bodies, and karyotype stability assessed by analyzing copy number variations by shallow DNA sequencing. Furthermore, a low-cost, decellularized ECM produced by human neonatal dermal fibroblasts was tested. In addition, all alternative coatings were tested for hiPSC differentiation into renal podocyte-like cells in a genome-wide transcriptomics screen. Our results show that all tested coatings were highly comparable to animal-derived Matrigel for both hiPSC maintenance and differentiation into renal podocyte-like cells. Furthermore, decellularized fibroblast-ECM could be a novel, attractive low-cost coating material.

Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro.

Adverse outcome pathways (AOPs) are organized sequences of key events (KEs) that are triggered by a xenobiotic-induced molecular initiating event (MIE) and summit in an adverse outcome (AO) relevant to human or ecological health. The AOP framework causally connects toxicological mechanistic information with apical endpoints for application in regulatory sciences. AOPs are very useful to link endophenotypic, cellular endpoints in vitro to adverse health effects in vivo. In the field of in vitro developmental neurotoxicity (DNT), such cellular endpoints can be assessed using the human "Neurosphere Assay," which depicts different endophenotypes for a broad variety of neurodevelopmental KEs. Combining this model with large-scale transcriptomics, we evaluated DNT hazards of two selected Chinese herbal medicines (CHMs) Lei Gong Teng (LGT) and Tian Ma (TM), and provided further insight into their modes-of-action (MoA). LGT disrupted hNPC migration eliciting an exceptional migration endophenotype. Time-lapse microscopy and intervention studies indicated that LGT disturbs laminin-dependent cell adhesion. TM impaired oligodendrocyte differentiation in human but not rat NPCs and activated a gene expression network related to oxidative stress. The LGT results supported a previously published AOP on radial glia cell adhesion due to interference with integrin-laminin binding, while the results of TM exposure were incorporated into a novel putative, stressor-based AOP. This study demonstrates that the combination of phenotypic and transcriptomic analyses is a powerful tool to elucidate compounds' MoA and incorporate the results into novel or existing AOPs for a better perception of the DNT hazard in a regulatory context.