npj Regenerative Medicine最新文献

The capacity of articular cartilage for self-repair is limited. Therefore, wide-ranging cartilage damage rarely resolves spontaneously, leading to the development of osteoarthritis. Previously, we developed human-induced pluripotent stem cell (hiPSC)-derived expandable human limb-bud-like mesenchymal (ExpLBM) cells with stable expansion and high chondrogenic capacity. In this study, various forms of articular cartilage-like tissue were fabricated using ExpLBM technology and evaluated to examine their potential as biomaterials. ExpLBM cells derived from hiPSCs were used to produce particle-like cartilage tissue and plate-like cartilage tissue. The cartilaginous particles and cartilaginous plates were transplanted into a minipig osteochondral defect model, and cartilage engraftment was histologically evaluated. For both transplanted cartilaginous particles and cartilaginous plates, good Safranin O staining and integration with the surrounding tissue were observed. Cartilaginous particles and cartilaginous plates made using hiPSCs-derived ExpLBM cells are effective for the regeneration of cartilage after injury.

Kidney organoids derived from human induced pluripotent stem cells lack a proper vasculature, hampering their applicability. Transplantation prevents the loss of organoid endothelial cells (ECs) observed in vitro, and promotes vascularization. In this study, we transplanted kidney organoids in chicken embryos and deployed single-cell RNA sequencing of ~12,000 organoid ECs to delineate their molecular landscape and identify key changes associated with transplantation. Transplantation significantly altered EC phenotypic composition. Consistent with angiogenesis, proliferating EC populations expanded 8 days after transplantation. Importantly, ECs underwent a major vein-to-arterial phenotypic shift. One of the transplantation-specific arterial EC populations, characterized by laminar shear stress response and Notch signalling, showed a similar transcriptome as human fetal kidney arterial/afferent arteriolar ECs. Consistently, transplantation-induced transcriptional changes involved proangiogenic and arteriogenic SOX7 transcription factor upregulation and regulon enrichment. These findings point to blood flow and candidate transcription factors such as SOX7 as possible targets to enhance kidney organoid vascularization.

Direct reprogramming is a breakthrough technology that can alter the fate of cells without the passage of stem cells. However, direct reprogramming of somatic cells into pulmonary alveolar epithelial cells has not yet been achieved. Here, we report the direct reprogramming of mouse tail tips and embryonic fibroblasts into induced pulmonary alveolar epithelial-like cells (iPULs) using four transcription factor-coding genes (Nkx2-1, Foxa1, Foxa2, and Gata6) and three-dimensional culture. The iPULs showed lamellar body-like structures and displayed key properties of pulmonary alveolar epithelial cells. Although the potential for iPULs to morphologically differentiate into alveolar epithelial type 1 cells was limited in vitro, the intratracheal administration of iPULs in a bleomycin-induced mouse model of pulmonary fibrosis led to their integration into the alveolar surface, where they formed both alveolar epithelial type 1 and type 2-like cells. Thus, reprogrammed fibroblasts may represent a new source of pulmonary alveolar epithelial cells for regenerative medicine.

Neuroma following nerve injury and/or amputation is a debilitating condition with significant impacts on quality of life. Several approaches exist to prevent or treat neuroma and/or reduce associated pain; however, these approaches are not consistently effective, facile, or widely accessible. The present study characterizes a xenogeneic nerve cap graft device (NCGD) composed of decellularized porcine nerve. The NCGD was assessed for its ability to inhibit nerve growth, neuroma formation, and pain in rodent models of sciatic neurectomy and tibial neuroma transposition. The NCGD provided a neuroinhibitory substrate that abated and interrupted nerve growth within 5 mm of the nerve stump and was progressively remodeled into healthy host-derived tissue. The NCGD also resulted in a 3.5-fold reduction in evoked pain and a decrease in pain-associated markers at the dorsal root ganglia. These results suggest that the NCGD may provide a simple and widely accessible alternative for prophylactic treatment of symptomatic neuroma.

Human skin is repaired by scar formation, lacking hair follicles, arrector pili muscles, and targeted innervation. Scarring leads to significant losses in skin functionality. Contrary to humans, spiny mice (Acomys spp.) repair skin via scar-free regeneration, regrowing hair follicles and muscles. However, skin across the body is diverse, and whether Acomys can regenerate specialized skin remains unclear. Here, we report that Acomys regenerated whisker pad skin with whisker follicles (i.e., vibrissae), blood sinuses, sebaceous glands, skeletal muscles, and targeted innervation. In contrast, CD-1 mice (Mus) healed via scarring and poor innervation of the scar. While whisker pad skin regeneration in Acomys was remarkable, only 20% of whiskers regenerated on average, ranging from 0% to 75%. Regenerated axons were bundled in epineurial sheaths, targeting the regenerated whisker, with an average of 75% of the uninjured innervation. This expands our understanding of Acomys skin regeneration and provides novel models for skin regeneration and sensorimotor recovery.

Inflammatory cells are crucial regulators of infection and regeneration that actively migrate to affected tissues. NF-kB and TNF-alpha (TNFα) are master regulators of immune signalling, but their importance for immune cell migration is much less well understood. We have therefore investigated how NF-kB and TNFα regulate both macrophage function and behaviour in vivo using a zebrafish model of tissue repair. We show that NF-kB activity differentially regulates TNFα activity through Tnf receptors 1a and 1b to control macrophage responses to injury. Loss of NF-kB in macrophages results in elevated TNFα expression and results in more directional migration. Impaired NF-kB activity in macrophages perturbs tissue regeneration, causes increased proliferation, altered pro- and anti-inflammatory gene expression and delays fin regeneration. We identify a crucial role for NF-kB modulation of TNFα signaling to regulate macrophage responses to tissue injury, which are necessary for effective fin regeneration.

In the United States, impaired bone healing impacts ~600,000 patients annually. Bone morphogenetic protein 2 (rhBMP2) therapy is impeded by low bone quality and adverse effects. Here, mesenchymal stem cells, engineered to produce BMP2 and BMP2/7 containing extracellular vesicles (BMP2-EV and BMP2/7-EV), provided an alternative means of stimulating bone formation. BMP2-EV and BMP2/7-EV drove increased calcium deposition and alkaline phosphatase activity; with increase in osterix, RUNX2, osteocalcin, and osteopontin documenting osteoblast differentiation. BMP2/7-EV induced SMAD phosphorylation and calcium deposition, was inhibited by DMH1, a BMP I receptor inhibitor, demonstrating BMP receptor dependence. BMP2 and BMP7 extracellular vesicle encapsulation was confirmed with preserved potency following treatment with BMP antagonist, Noggin. Application of BMP2/7-EV in a rat calvarial defect model demonstrated enhanced bone formation on micro-computed tomography and histopathologic analysis, equaling rhBMP2. BMP2/7-EV mediated bone formation here highlights EVs as a unique modality for delivery of tailored polyvalent regenerative biotherapies.

Hippo-Yap/Taz pathway is essential for tissue regeneration in multiple species. However, we found that in the highly regenerative salamanders, Yap knockout does not compromise the limb regeneration due to genetic compensation response (GCR). Specifically, the mutated Yap locus derived non-sense mRNA, which was recognized by UPF3A to instruct compensatory Taz induction. Blocking Yap mRNA or protein indeed inhibits regeneration. GCR could be utilized to maintain the robustness of limb regeneration.

Advanced protocols show potential for human stem cells (SC)-derived islets generation under planar (2D) alone or three-dimensional (3D) cultures, but show challenges in scalability, cell loss, and batch-to-batch consistency. This study explores Vertical Wheel (VW)® bioreactor suspension technology to differentiate islets from human induced pluripotent stem cells, achieving uniform, transcriptionally mature, and functional SC-islets. A 5x increase in scale from 0.1 L to 0.5 L reactors resulted in a 12-fold (15,005-183,002) increase in islet equivalent count (IEQ) without compromising islet structure. SC-islets show enriched β-cell composition (~63% CPPT⁺NKX6.1⁺ISL1⁺), glucose responsive insulin release (3.9-6.1-fold increase), and reversed diabetes in STZ-treated mice. Single cell RNA sequencing and flowcytometry analysis confirmed transcriptional maturity and functional identity, similar to adult islets. Lastly, harvested SC-islet grafts demonstrate improved islet functionality and mature transcriptomic signatures. Overall, scale-up in VW® bioreactor technology enhances IEQ yield with minimal variability and reduced cell loss, offering a pathway for clinical-grade SC-islet production.

The salivary gland (SG) is vital for oral function and overall health through secretion of saliva. However salivary dysfunction due to aging, medications, autoimmune disorders, and cancer treatments poses significant challenges. We established the first diverse and clinically annotated salivary regenerative biobank at Mayo Clinic to study salivary gland stem/progenitor cells (SGSPCs). Optimization of cell isolation and progenitor assays revealed SGSPCs enriched within the CD24/EpCAM/CD49f+ and PSMA- phenotypes of both submandibular and parotid glands, with clonal differentiation assays highlighting heterogeneity. Induction of PSMA/FOLH1 expression was associated with SGSPC differentiation. Using mass spectrometry-based single cell proteomics, we identified 2461 proteins in SGSPC-enriched cells, including co-expressed cytokeratins, expressed in rare salivary ductal basal cells. Additionally, PRDX, a unique class of peroxiredoxin peroxidases enriched in SGSPCs, demonstrated H₂O₂-dependent growth, suggesting a role in salivary homeostasis. These findings provide a foundation for SGSPC research and potential regenerative therapies for salivary gland dysfunction.