Diabetes mellitus (DM) is a metabolic disease characterized by persistent hyperglycemia, resulting from defects in insulin secretion or impaired insulin action. In cases of severe pancreatic cell dysfunction and deficiency, the primary treatment remains lifelong insulin injections. A potential alternative is allogeneic pancreatic cell transplantation from a donor, which can stabilize glucose levels. However, the scarcity of donor material and the risk of immune rejection limit the widespread use of this approach. An alternative solution involves using in vitro-derived insulin-producing cells generated through the differentiation of pluripotent stem cells (PSCs), which could overcome the shortage of transplantable material. Furthermore, patient-specific cells—obtained directly from the patient via reprogramming of blood or skin cells into induced pluripotent stem cells (iPSCs)—would avoid immune rejection. Advances in this field have led to the active development and optimization of PSC differentiation into hormone-producing cells worldwide, with more than hundred patients dosed in clinical trials with ESC-derived cells and the single trial of iPSC-derived cells. This review highlights recent progress and prospects in generating insulin-producing cells from human PSCs, their applications in therapy development and disease modeling, as well as the current challenges and potential solutions.

Today, the cell-based technologies are one of the instruments used for the cartilage tissue repair. Creation of a universal hypoimmunogenic cartilage tissue graft from the differentiated derivatives of induced pluripotent stem cells (iPSCs) might solve the problem of the lack of the cartilage cell product. However, currently there is little data on immunogenicity of such tissue-engineered preparations. The study was aimed to create a cartilage implant from the differentiated derivatives of the B2M-deficient iPSCs and assess its immunogenicity. The previously developed protocol was used to ensure differentiation of both wild-type and B2M knockout iPSCs into chondrocyte-like cells. After quality control of the resulting cell lines by conducting polymerase chain reaction and immunocytochemical assessment, the resulting cell lines were co-cultured with the peripheral blood mononuclear cells of a healthy donor. When co-cultivation was over, activation and degranulation of CD8+ T cells was assessed by flow cytometry analysis based on the CD69 and CD107a expression on the cell surface, respectively. The iPSC-derived chondrocytes expressed the cartilage tissue markers. Flow cytometry analysis revealed no substantial differences in immunogenicity between the derivatives of wild-type and B2M knockout iPSCs, as well as from the cartilage tissue cells of a healthy donor. Immunogenicity of chondrocyte-like cells was higher than that of hypoimmunogenic non-edited iPSCs. The B2M knockout iPSCs demonstrated a trend towards greater activation of CD8+ T cells. Thus, the B2M knockout in the iPSC-derived chondrocytes had no significant effect on the tissue immunogenicity. It is necessary to further edit the genes encoding MHC II and CD47 to obtain a less immunogenic product.

BACKGROUND: Cryopreservation is a widely used method for long-term viability preservation of cultured cells and complex cellular structures, including organoids, which serve both scientific and clinical purposes, as well as for screening applications. However, data on cryopreservation of organoids derived from differentiated induced pluripotent stem cells (iPSCs) remain limited. AIM: The work aimed to optimize slow freeze protocols for neural organoids derived from iPSC-differentiated cells and chondrospheres. METHODS: Neural organoids differentiated from iPSCs were cryopreserved on days 9, 14, 22, 29, and 43 using four different cryopreservation media. Post-thaw assessment included organoid integrity and size measurements, quantitative polymerase chain reaction for MAP2 and NES markers, and immunohistochemical (IHC) staining for βIII-tubulin, MAP2, SOX2, and proliferating cell nuclear antigen (PCNA). Chondrospheres were derived from human chondrocytes and cryopreserved 29 days after transition to 3D culture conditions in a solution of 82% DMEM + 10% FBS + 8% DMSO + 10 µM ROCK inhibitor Y27632 (Ri, Rho-associated protein kinase inhibitor). Post-thaw IHC analysis was performed to assess the expression of chondrogenic marker proteins, including aggrecan, collagen type II, and SOX9, as well as PCNA. RESULTS: Neural organoids cryopreserved between weeks 3 and 6 of differentiation retained the highest post-thaw structural integrity. Although organoid diameter decreased by 14.5% on average 1 week after thawing, size was nearly restored to the values observed on the corresponding day of differentiation for organoids not subjected to cryopreservation by week 2. Quantitative polymerase chain reaction and IHC staining confirmed maintenance of neuronal phenotype 2 weeks post-thaw. Chondrospheres showed no change in diameter over 2 weeks post-thaw and exhibited 100% preservation of organoid integrity. IHC analysis confirmed the presence of chondrocytic proteins in chondrospheres 2 weeks after thawing. CONCLUSION: Based on the study findings, the third week of differentiation was identified as the optimal stage for cryopreservation of iPSC-derived neural organoids. The cryopreservation medium composed of 82% DMEM, 10% FBS, and 8% DMSO was optimal for both neural organoids and chondrospheres.

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

BACKGROUND: Chronic joint diseases represent a significant medical and social problem due to their high prevalence, frequent patient disability, and associated economic losses. Potential therapeutic approaches include cell-based technologies utilizing autologous chondrocytes, which are surgically harvested and expanded in vitro before transplantation. Recently, the 3D chondrocyte culturing technique in the form of spheroids has been increasingly used, as it better preserves the functional state of chondrocytes and creates favorable conditions for high-quality cartilage maturation. However, the quantity and quality of autologous chondrocytes may be insufficient to generate an adequate amount of cellular material to fully repair large articular cartilage defects. An alternative cell source could be chondrocytes derived from induced pluripotent stem cells. AIM: To perform an immunophenotypic comparison of spheroids composed of native chondrocytes and spheroids of chondrocytes differentiated from human induced pluripotent stem cells. MATERIALS AND METHODS: This study presents detailed protocols for generating spheroids of native chondrocytes and spheroids of induced pluripotent stem cells–derived chondrocytes using mini-bioreactors and recombinant TGF-β1 and BMP2 factors. Using immunocytochemical staining and quantitative reverse transcription polymerase chain reaction, we compared native chondrocyte spheroids and spheroids of chondrocytes differentiated from induced pluripotent stem cells in terms of the expression of key chondrogenic genes: aggrecan (ACAN), collagen types I (COL1A2) and II (COL2A1), and the key transcription factor SOX9. RESULTS: The expression levels of chondrogenic genes in induced pluripotent stem cells–derived chondrocyte spheroids closely resembled those in native chondrocyte spheroids, except for a significantly increased expression of type I collagen (COL1A2). CONCLUSION: The use of induced pluripotent stem cells–derived chondrocyte spheroids represents a promising approach for the treatment of chronic joint diseases.