Introduction Tendon injuries heal through disorganized extracellular matrix (ECM) deposition rather than the restoration of native collagen architecture, thus resulting in suboptimal functional recovery. Although adipose-derived stem cells (ADSCs) are attractive for tendon regeneration because of their accessibility and expandability, their tenogenic differentiation efficiency is lower than that of tendon-derived stem cells (TDSCs). C1q/TNF-related protein 3 (CTRP3) is an adipokine implicated in ECM remodeling and fibrosis; however, its role in tendon lineage regulation is unknown. Objective CTRP3 (C1q/TNF-related protein 3) contributes to extracellular-matrix remodeling and, in tendon, is associated with a pathological fibrotic matrix signature; we therefore reasoned that reducing CTRP3 would favor a more mature tendon-like matrix. Using a loss-of-function approach based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), we investigated whether CTRP3 disruption alters tenogenic marker expression, at both the transcript and protein levels, and the tendon-associated ECM marker profile of canine ADSCs relative to wild-type ADSCs and native TDSCs. Methods CRISPR/Cas9-mediated CTRP3–disrupted ADSCs were generated and validated using sequencing, quantitative polymerase chain reaction, and Western blotting. Wild-type ADSCs, CTRP3-disrupted ADSCs, and TDSCs were subjected to tenogenic induction for 0–3 weeks. Tenogenic transcriptional and ECM markers were analyzed at mRNA and protein levels. Results CTRP3-disrupted ADSCs showed increased collagen type I and tenomodulin (TNMD) expression during tenogenic induction compared with wild-type ADSCs. COL III expression showed a transient increase followed by a decline, with lower expression in CTRP3-disrupted cADSCs than in WT cADSCs at week 2. CTRP3 disruption increased early Mkx and Scx expression at the earliest time points but did not consistently alter the early transcriptional program, which rose comparably across all groups. CTRP3-disrupted cADSCs showed partial, marker- and time-point-dependent overlap with native cTDSCs, mainly for selected late ECM markers rather than across the overall transcript and protein profiles. Conclusion CTRP3 disruption shifted the tendon-associated ECM marker profile during the tenogenic differentiation of ADSCs, with reduced COL III accumulation, rather than uniformly enhancing tenogenic differentiation. Although early transcriptional changes were observed, the more consistent differences were found in late tendon-associated ECM markers. The modulation of CTRP3 may represent a candidate strategy worth further investigation for stem cell–based tendon regeneration in veterinary medicine.
Introduction Decompensated cirrhosis is associated with a poor prognosis and limited therapeutic options for patients who are not candidates for liver transplantation. Although various cell-based regenerative therapies have been investigated, their clinical benefits are limited. Autologous chemically-induced liver progenitor cells (CLiPs) are expandable progenitor-like cells generated from mature hepatocytes using small-molecule reprogramming and have demonstrated regenerative potential in multiple preclinical models. Because CLiPs are bipotent and can differentiate toward both hepatocytic and biliary epithelial lineages, their lineage fate after transplantation may be influenced by the recipient liver microenvironment, and whether transplanted CLiPs undergo hepatocytic maturation in the human decompensated cirrhotic liver to the extent observed in animal models remains unknown. We describe the rationale and design of the first-in-human clinical trial evaluating autologous CLiP transplantation for decompensated cirrhosis. Setting Autologous CLiP cells (CLP-001) are manufactured from liver tissue obtained by laparoscopic partial hepatectomy and expanded in a certified Cell Processing Center under controlled conditions. Quality and safety assessment includes histopathological examination of the harvested liver tissue, sterility, endotoxin and mycoplasma testing, viable cell count, cell viability, cell-surface marker characterization, copy-number variation (CNV) analysis, and targeted cancer-related gene-panel testing (Table 1). The study is conducted under the Japanese Act on the Safety of Regenerative Medicine and has been approved by a Certified Special Committee for Regenerative Medicine. Methods This is a first-in-human, single-center, open-label, single-arm phase I/II study. Three adults with Child–Pugh class B decompensated cirrhosis who are not candidates for liver transplantation will be enrolled. Approximately 30 g of liver tissue will be harvested laparoscopically and used for CLiP manufacturing over a 12–16 week period. Following successful quality and safety testing, CLP-001 will be administered into the portal venous system through surgical cannulation of the ileocolic vein. The primary endpoint is safety, defined as adverse events and product-related deficiencies from tissue procurement through 1 month after transplantation. Secondary and exploratory endpoints include safety through 12 months and descriptive changes in liver function, fibrosis-related biomarkers, liver stiffness, and clinical manifestations of cirrhosis. Conclusion This is, to our knowledge, the first clinical evaluation of autologous CLiP transplantation in patients with decompensated cirrhosis. The trial is designed to establish the safety and procedural feasibility of the entire therapeutic workflow, including tissue procurement, cell manufacturing, quality assurance, and portal venous administration. Efficacy-related observations are exploratory, and the lineage fate of transplanted CLiPs cannot be directly established in this study. The findings are expected to provide the first clinical evidence regarding the safety and feasibility of autologous CLiP transplantation and support the future development of CLiP-based regenerative therapy for advanced chronic liver disease. At the time of submission, patient enrollment has commenced, and no participant has yet undergone CLiP transplantation.
Among different types of arthritis, osteoarthritis (OA) and rheumatoid arthritis (RA) remain the most common and primary cause of pain and disability worldwide, characterized by progressive joint degeneration and persistent inflammation. Although there have been developments in pharmacology, the available treatment program slows disease progression and provides only symptomatic relief. However, their ability to repair and restore damaged tissue is limited. This highlights the importance of regenerative therapy in actively modulating the immune system while stimulating tissue regeneration. We conducted a narrative review of PubMed, Scopus, Web of science, ClinicalTrials.gov, and ChiCTR, focusing on preclinical and clinical studies of umbilical cord-derived mesenchymal stem cells (UC-MSCs) therapy in OA and RA. Recently, UC-MSC have been emerging as a promising therapeutic approach due to their immunomodulatory and regenerative properties. Because they are harvested noninvasively, these cells have greater proliferative capacity and lower immunogenicity than MSCs derived from bone marrow or adipose tissue. UC-MSCs do not replace damaged cells; instead, they signal other cells to produce bioactive substances that reduce inflammation and promote cartilage repair. Data from emerging preclinical and clinical trials, which show significant improvements in WOMAC and VAS scores and a decrease in DAS28 with minimal adverse effects, suggest that UC-MSCs will improve pain and function and, in turn, overall quality of life. UC-MSCs represent a mechanistically distinct and clinically promising therapeutic effect for OA and RA, addressing both the inflammatory and degenerative condition of joint disease. However, there are still large, well-designed, standardized clinical trials that can best optimize doses and treatment plans. This narrative review summarizes the various forms of arthritis, disease pathogenesis, and preclinical and clinical evidence for the use of UC-MSCs in the treatment of arthritis, such as OA and RA.
Introduction The recombinant laminin-511 E8 fragment (LM511E8) is a highly effective, xeno-free matrix for human pluripotent stem cell (hPSC) culture. However, the firm adhesion of hPSCs to LM511E8 conventionally requires mechanical scraping after enzymatic treatment, thereby compromising cell viability, reproducibility, and culture scalability. Methods We developed a step-minimized, ethylenediaminetetraacetic acid (EDTA)-based, scraper-free detachment protocol for hPSCs cultured on LM511E8. The protocol eliminates both mechanical scraping and centrifugation, and was evaluated for detachment efficiency, long-term maintenance of hPSC quality, applicability to mid-scale culture systems, and the effects of trypsin-like proteases on EDTA-mediated cell detachment. Results The protocol achieved consistently high detachment efficiency with minimal handling while preserving hPSC pluripotency and genomic integrity over long-term culture. Notably, the addition of trypsin-like proteases to the EDTA solution reduced detachment efficiency, which was restored by protease inhibition. Furthermore, the method was readily adaptable to mid-scale culture systems (e.g., multilayer flasks) with minor modifications. Conclusions Our protocol provides an efficient, reproducible, and centrifugation-free detachment method that resolves the technical limitations of LM511E8-based cultures. This scalable approach may facilitate high-throughput hPSC expansion in both basic research and regenerative medicine applications.
Introduction Teriparatide (TPTD) is an established osteoanabolic agent for the treatment of osteoporosis; however, its short circulating half-life necessitates repeated administration, while limited skeletal selectivity may reduce delivery efficiency. To address these limitations, we developed an Asp6-functionalized, bone-targeting exosome system carrying TPTD mRNA (Asp6-Exo@TPTD). An Exo-Motif sequence was incorporated into the TPTD mRNA construct to facilitate hnRNPA2B1-associated enrichment of the transcript in exosomes, while surface modification with the hydroxyapatite-binding peptide Asp6 was used to enhance skeletal affinity. BMSC-derived exosomes may additionally provide intrinsic regenerative signals that complement the osteoanabolic activity of TPTD. Methods Mouse bone marrow mesenchymal stromal cells (BMSCs) were incubated with different exosome formulations to evaluate intracellular TPTD mRNA transfer, TPTD protein production, CRE-dependent reporter activity, and osteogenic differentiation. Free TPTD was included as a pharmacological reference in the functional, osteogenic, and animal efficacy experiments. The bone-binding properties of the engineered exosomes were assessed using hydroxyapatite-containing bone samples and ex vivo bone tissues. In vivo biodistribution was evaluated by fluorescence imaging after intravenous administration. Therapeutic efficacy was investigated in an ovariectomy-induced osteoporosis mouse model by assessing trabecular microarchitecture, histological changes, serum bone-formation markers, and dynamic bone formation. Preliminary tolerability was evaluated by major-organ histology and serum biochemical analyses, including serum calcium and hepatic and renal function indices. Results Asp6-Exo@TPTD increased intracellular TPTD mRNA levels and TPTD secretion in recipient BMSCs and enhanced CRE-dependent luciferase activity, osteogenic gene expression, alkaline phosphatase staining, and extracellular matrix mineralization. Asp6-Exo@TPTD exhibited greater binding to bone tissues than both unloaded Exo and non-targeted Exo@TPTD in vitro and ex vivo. Following systemic administration, bone-associated fluorescence remained detectable for at least 72 h, with greater femoral and tibial fluorescence and lower relative hepatic fluorescence than those observed with unmodified exosomes. In ovariectomized mice, weekly administration of Asp6-Exo@TPTD improved trabecular microarchitecture, increased serum procollagen type I N-terminal propeptide levels, and enhanced the mineral apposition rate. No overt histopathological abnormalities or significant alterations in serum calcium or routine hepatic and renal biochemical indices were detected. Conclusions Asp6-functionalized exosomes carrying TPTD mRNA promoted osteogenic activity, enhanced bone-associated distribution, and attenuated ovariectomy-induced bone loss. These findings support Asp6-Exo@TPTD as a promising exosome-based strategy for bone-targeted TPTD mRNA delivery and osteoporosis treatment.
