Makoto Kondo

Background: Regeneration of damaged cartilage is challenging, and no reproducible regenerative therapies using mass-producible cell products have been established. This study evaluated the cartilage regeneration capability and therapeutic scalability using cell sheets derived from routinely available surgical waste cartilage tissues of young and adult patients, while also investigating the mechanisms that define the characteristics of each cell type. Methods:We compared the viability, proliferation, and cell sheet characteristics of juvenile cartilage-derived chondrocytes (JCCs) from patients with polydactyly (2.2 ± 1.6 years) and adult cartilage-derived chondrocytes (ACCs) from patients with femoroacetabular impingement (FAI) (34.1 ± 10.6 years)in vitro. Thein vivocartilage regeneration capability of each cell sheet was validated in a nude rat knee cartilage defect model using histological O’Driscoll score evaluation on Safranin-O-stained tissues and immunohistochemistry. JCC sheets (n = 13) and ACC sheets (n = 8) were analyzed using established bulk RNA sequencing pipelines for gene ontology (GO) analysis, gene set enrichment analysis (GSEA), and ingenuity pathway analysis (IPA). Interferon gamma (IFN-γ) was applied to JCC sheet culture for confirmation of the interferon signaling involvement in cell proliferation, cell sheet characteristics, and chondrogenic differentiation.Results:JCC demonstrated higher colony-forming ability and stable high proliferation compared to ACC. Both JCC and ACC sheets formed positively stained hyaline cartilage for Safranin-O, type II collagen, aggrecan, and human vimentin, while being negative for type I collagen, four weeks after rat transplantation. However, the regenerated cartilage from ACC sheet transplantation was found to be thinner compared to that from JCC sheet transplantation. Comprehensive gene analysis revealed significant activation of IFN signaling in the ACC sheets. Furthermore, the addition of exogenous IFN dramatically reduced the proliferation and cartilage formation capability of JCC.Conclusions:JCC sheets exhibit high therapeutic scalability due to their proliferation and cartilage regeneration capabilities presumably derived from sustained low IFN-γ activity. Consideration of the donor age and tissue inflammatory status is essential for the cell source in allogeneic cell therapies. Given their sustainable sourcing from routine surgical discards, JCCs present a commercially viable and scalable option for allogeneic regenerative therapy in cartilage repair.

Purpose: This study aimed to establish a combined histological assessment system of neo-cartilage outcomes and to evaluate variations in an established rat defect model treated with human juvenile cartilage-derived chondrocyte (JCC) sheets fabricated from various donors. Methods: JCCs were isolated from the polydactylous digits of eight patients. Passage 2 (P2) JCC sheets from all donors were transplanted into nude rat chondral defects for 4 weeks (27 nude rats in total). Defect-only group served as control. Histological samples were stained for safranin O, collagen 1 (COL1), and collagen 2 (COL2). (1) All samples were scored, and correlation coefficients for each score were calculated. (2) Donors were divided into “more effective” and “less effective” groups based on these scores. Then, differences between each group in each category of modified O’Driscoll scoring were evaluated. Results: (1) Modified O’Driscoll scores were negatively correlated with %COL1 area, and positively correlated with %COL2 area and COL2/1 ratio. (2) Four of 8 donors exhibited significantly higher modified O’Driscoll scores and %COL2 areas. JCC donors were divided into two groups by average score values. Significant differences between the two groups were observed in modified O’Driscoll categories of “Nature of predominant tissue,” “Reconstruction of subchondral bone,” and “Safranin O staining.” Conclusion: The combined histological evaluation method is useful for detailed in vivo efficacy assessments of cartilage defect regeneration models. Variations in histological scores among juvenile cartilage-derived chondrocyte donors were correlated to the quality of regenerated cartilage hyaline structure and subchondral bone remodeling observed in the nude rat defect model.

Leukemia cutis or leukemic cell infiltration in skin is one of the common extramedullary manifestations of acute myeloid leukemia (AML) and signifies a poorer prognosis. However, its pathogenesis and maintenance remain understudied. Here, we report massive AML cell infiltration in the skin in a transplantation-induced MLL-AF9 AML mouse model. These AML cells could regenerate AML after transplantation. Prospective niche characterization revealed that skin harbored mesenchymal progenitor cells (MPCs) with a similar phenotype as BM mesenchymal stem cells. These skin MPCs protected AML-initiating stem cells (LSCs) from chemotherapy in vitro partially via mitochondrial transfer. Furthermore, Lama4 deletion in skin MPCs promoted AML LSC proliferation and chemoresistance. Importantly, more chemoresistant AML LSCs appeared to be retained in Lama4−/− mouse skin after cytarabine treatment. Our study reveals the characteristics and previously unrecognized roles of skin mesenchymal niches in maintaining and protecting AML LSCs during chemotherapy, meriting future exploration of their impact on AML relapse.

Background Scaffold-free cell-dense implants, termed “cell sheets”, are routinely fabricated using temperature-responsive commercial cell culture surfaces. In vivo regenerative capacity of human juvenile cartilage-derived chondrocyte (JCC) sheets is reported in both human clinical studies and nude rat defect models. How human cell source variables affect sheet tissue regeneration remains unelucidated. Purpose To evaluate the variation of detailed histological neo-cartilage outcomes in an established rat defect model treated with human JCC sheets fabricated from various donors and different JCC passages. Study Design Descriptive laboratory study. Methods JCCs were isolated from 8 amputated human polydactylous digits. Passage 2 (P2) JCC sheets from all donors and P9 JCC sheets from one donor were transplanted into nude rat chondral defects for 4 weeks. Defect-only group served as control. Histological samples were stained for safranin-O, collagen 1 (COL1), and collagen 2 (COL2). Modified O’Driscoll scores, %COL1 and %COL2 areas, and COL2/1 ratios were applied for histological semi-quantitative evaluations. (1) All samples were scored and correlation coefficients for each score calculated. (2) Donors treated with P2 JCC sheets were divided into “more effective” and “less effective” groups based on these scores. Then, differences between each group in each category of modified O’Driscoll scoring were evaluated. (3) Scores of P2 and P9 samples from the same JCC donor were compared. Results (1) Mean modified O’Driscoll score, IHC %COL1 and COL2 areas, and COL2/1 ratio was 20.7±3.8, 37.9±27.5, 63.1±30.1, and 3.9±4.0, respectively. Modified O’Driscoll scores were negatively correlated with %COL1 area, and positively correlated with %COL2 area and COL2/1 ratio. (2) Four of 8 donors exhibited significantly higher modified O’Driscoll scores and %COL2 areas compared to the defect-only control group. JCC donors were divided into two groups by average score values: groups 3, 4, 6, and 7 (group ‘more effective’) and groups 1, 2, 5, and 8 (group less effective’). Significant differences between the two groups were observed in Modified O’Driscoll categories of “Nature of predominant”, “Reconstruction of subchondral bone”, and “Safranin-O staining”. (3) Modified O’Driscoll scores and COL2/1 ratio for JCC P2 were significantly higher than that from P9. Conclusion The combined histological evaluation method employed is useful for detailed in vivo efficacy assessments of cartilage defect regeneration models. The analysis demonstrated that regenerative effects with JCC sheets varied both by JCC donor and passage number. Variations in histological evaluations among JCC donors were correlated to the quality of regenerated cartilage hyaline structure and subchondral bone remodeling observed in the nude rat defect model. Clinical Relevance This study provides a useful screening and prediction algorithm for cartilage regenerative success from human cell-derived implants in a rodent defect model. What is known about the subject Human juvenile cartilage-derived chondrocyte (JCC) sheets have shown safety and cartilage regenerative capacity in both nude rat chondral defects and a recent limited allogenic human clinical research study. Allogenic cell source variability is known to affect cell-based cartilage regeneration outcomes. Few predictive correlates exist to predict cell source regenerative success. Both Passage 2 (P2) and Passage 9 (P9) JCC-derived chondrogenic pellets exhibit strong characteristic safranin-O staining, but cultured P9 cultured pellet size is smaller than that of P2 pellets. What this study adds to existing knowledge Cartilage regenerative effects vary highly with banked cell source. Current cartilage regenerative strategies in preclinical models do not predict human performance and are clinically unreliable; few off-the-shelf, scalable “living” options are available. Cartilage regeneration using banked human JCC sheets represents a scalable, allogenic, scaffold-free approach now successfully tested both in nude rat and human chondral defects. This study shows that histological evaluation of sheets from several JCC donor demonstrated variable regenerated cartilage hyaline structure and subchondral bone remodeling in a nude rat across donor and passage. Tools to better predict cell-based cartilage regenerative reliability by screening banked allogenic human cell sources in a rat model are described.

Although tyrosine kinase inhibitors are effective for treating chronic myeloid leukemia (CML), they often fail to eradicate the leukemia-initiating stem cells (LSCs), causing disease persistence and relapse. Evidence indicates that LSC persistence may be due to bone marrow (BM) niche protection. However, little is known about the underlying mechanisms. We here molecularly and functionally characterized BM niches in CML patients at diagnosis and revealed the altered niche composition and function in the CML patients. Long-term culture initiating cell (LTC-IC) assay showed that the mesenchymal stem cells from CML patients displayed an enhanced supporting capacity for normal and CML BM CD34+CD38- cells. Molecularly, RNA sequencing detected dysregulated cytokine and growth factor expression in CML patient BM cellular niches. Among them, CXCL14 was lost in the BM cellular niches in contrast to its expression in healthy BM. Restoring CXCL14 significantly inhibited CML LSC maintenance and enhanced their response to imatinib in vitro, and CML engraftment in vivo in NSG-SGM3 mice. Importantly, CXCL14 treatment dramatically inhibited CML engraftment in xenografted NSG-SGM3 mice, even to a greater degree than imatinib, and this inhibition persisted in patients with suboptimal TKI response. Mechanistically, CXCL14 upregulated inflammatory cytokine signaling but downregulated mTOR signaling and oxidative phosphorylation in CML LSCs. Together, we have discovered a suppressive role of CXCL14 in CML LSC growth. CXCL14 might offer a treatment option targeting CML LSCs.

A focal advantage of cell sheet technology has been as a scaffold-free three-dimensional (3D) cell delivery platform capable of sustained cell engraftment, survival, and reparative function. Recent evidence demonstrates that the intrinsic cell sheet 3D tissue-like microenvironment stimulates mesenchymal stem cell (MSC) paracrine factor production. In this capacity, cell sheets not only function as 3D cell delivery platforms, but also prime MSC therapeutic paracrine capacity. This study introduces a “cell sheet multilayering by centrifugation” strategy to non-invasively augment MSC paracrine factor production. Cell sheets fabricated by temperature-mediated harvest were first centrifuged as single layers using optimized conditions of rotational speed and time. Centrifugation enhanced cell physical and biochemical interactions related to intercellular communication and matrix interactions within the single cell sheet, upregulating MSC gene expression of connexin 43, integrin β1, and laminin α5. Single cell sheet centrifugation triggered MSC functional enhancement, secreting higher concentrations of pro-regenerative cytokines vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10). Subsequent cell sheet stacking, and centrifugation generated cohesive, bilayer MSC sheets within 2 h, which could not be accomplished within 24 h by conventional layering methods. Conventional layering led to H1F-1α upregulation and increased cell death, indicating a hypoxic thickness limitation to this approach. Comparing centrifuged single and bilayer cell sheets revealed that layering increased VEGF production 10-fold, attributed to intercellular interactions at the layered sheet interface. The “MSC sheet multilayering by centrifugation” strategy described herein generates a 3D MSC-delivery platform with boosted therapeutic factor production capacity.

Leukemia cutis or leukemic cell infiltration in skin is one of the common extramedullary manifestations of acute myeloid leukemia (AML) and signifies a poorer prognosis. However, its pathogenesis and maintenance remain understudied. Here, we report massive AML cell infiltration in the skin in a transplantation-induced MLL-AF9 AML mouse model. These AML cells could regenerate AML post-transplantation. Prospective niche characterization revealed that skin harbored mesenchymal progenitor cells (MPCs) with a similar phenotype as BM mesenchymal stem cells. These skin MPCs protected AML-initiating stem cells (LSCs) from chemotherapy in vitro partially via mitochondrial transfer. Furthermore, Lama4 deletion in skin MPCs promoted AML LSC proliferation and chemoresistance. Importantly, more chemoresistant AML LSCs appeared to be retained in Lama4 ^-/- mouse skin post-cytarabine treatment. Our study reveals the characteristics and previously unrecognized roles of skin mesenchymal niches in maintaining and protecting AML LSCs during chemotherapy, meriting future exploration of their impact on AML relapse. A 40-word summary Sandhow et al have in transplantation-induced AML mouse models demonstrated the leukemia-regenerating capacity of AML cells infiltrated in the skin and the role of skin mesenchymal niches in maintaining/protecting AML cells, providing new insight into the pathology of leukemia cutis.

Impairment of normal hematopoiesis and leukemia progression are 2 well-linked processes during leukemia development and are controlled by the bone marrow (BM) niche. Extracellular matrix proteins, including laminin, are important BM niche components. However, their role in hematopoiesis regeneration and leukemia is unknown. Laminin α4 (Lama4), a major receptor-binding chain of several laminins, is altered in BM niches in mice with acute myeloid leukemia (AML). So far, the impact of Lama4 on leukemia progression remains unknown. We here report that Lama4 deletion in mice resulted in impaired hematopoiesis regeneration following irradiation-induced stress, which is accompanied by altered BM niche composition and inflammation. Importantly, in a transplantation-induced MLL-AF9 AML mouse model, we demonstrate accelerated AML progression and relapse in Lama4−/− mice. Upon AML exposure, Lama4−/− mesenchymal stem cells (MSCs) exhibited dramatic molecular alterations, including upregulation of inflammatory cytokines that favor AML growth. Lama4−/− MSCs displayed increased antioxidant activities and promoted AML stem cell proliferation and chemoresistance to cytarabine, which was accompanied by increased mitochondrial transfer from the MSCs to AML cells and reduced reactive oxygen species in AML cells in vitro. Similarly, we detected lower levels of reactive oxygen species in AML cells from Lama4−/− mice post–cytarabine treatment. Notably, LAMA4 inhibition or knockdown in human MSCs promoted human AML cell proliferation and chemoprotection. Together, our study for the first time demonstrates the critical role of Lama4 in impeding AML progression and chemoresistance. Targeting Lama4 signaling pathways may offer potential new therapeutic options for AML.

Knee cartilage does not regenerate spontaneously after injury, and a gold standard regenerative treatment algorithm has not been established. This study demonstrates preclinical safety and efficacy of scaffold-free, human juvenile cartilage-derived-chondrocyte (JCC) sheets produced from routine surgical discards using thermo-responsive cultureware. JCCs exhibit stable and high growth potential in vitro over passage 10, supporting possibilities for scale-up to mass production for commercialization. JCC sheets contain highly viable, densely packed cells, show no anchorage-independent cell growth, express mesenchymal surface markers, and lack MHC II expression. In nude rat focal osteochondral defect models, stable neocartilage formation was observed at 4 weeks by JCC sheet transplantation without abnormal tissue growth over 24 weeks in contrast to the nontreatment group showing no spontaneous cartilage repair. Regenerated cartilage was safranin-O positive, contained type II collagen, aggrecan, and human vimentin, and lacked type I collagen, indicating that the hyaline-like neocartilage formed originates from transplanted JCC sheets rather than host-derived cells. This study demonstrates the safety of JCC sheets and stable hyaline cartilage formation with engineered JCC sheets utilizing a sustainable tissue supply. Cost-benefit and scaling issues for sheet fabrication and use support feasibility of this JCC sheet strategy in clinical cartilage repair.