Recent progress of animal transplantation studies for treating articular cartilage damage using pluripotent stem cells

Focal articular cartilage damage can eventually lead to the onset of osteoarthritis with degradation around healthy articular cartilage. Currently, there are no drugs available that effectively repair articular cartilage damage. Several surgical techniques exist and are expected to prevent progression to osteoarthritis, but they do not offer a long-term clinical solution. Recently, regenerative medicine approaches using human pluripotent stem cells (PSCs) have gained attention as new cell sources for therapeutic products. To translate PSCs to clinical application, appropriate cultures that produce large amounts of chondrocytes and hyaline cartilage are needed. So too are assays for the safety and efficacy of the cellular materials in preclinical studies including animal transplantation models. To confirm safety and efficacy, transplantation into the subcutaneous space and articular cartilage defects have been performed in animal models. All but one study we reviewed that transplanted PSC-derived cellular products into articular cartilage defects found safe and effective recovery. However, for most of those studies, the quality of the PSCs was not verified, and the evaluations were done with small animals over short observation periods. Large animals and longer observation times are preferred. We will discuss the recent progress and future direction of the animal transplantation studies for the treatment of focal articular cartilage damages using PSCs.This is the peer reviewed version of the following article: Yamashita A., Tsumaki N.. Recent progress of animal transplantation studies for treating articular cartilage damage using pluripotent stem cells. Development Growth and Differentiation 63, 72 (2021), which has been published in final form at https://doi.org/10.1111/dgd.12706. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited

Low Genetic Differentiation with High Genetic Variability Observed in Common Coastal Starfish Asterina pectinifera around Japan Inferred from Isozyme Analysis

Genetic variability and geographic population structure in population of the common coastal starfish Asterina pectinifera were examined in nine localities around Japan through isozyme analysis. Surveying 28 enzymes by using starch gel electrophoresis, it was found that eight loci controlling seven enzymes were useable as genetic markers. Genetic variability of this species estimated from the 8 loci followed ; the proportion of variant loci ranged from 0.63 to 1.00 with a mean of 0.80, expected average heterozygosity was from 0.176 to 0.208 with a mean of 0.184, and mean number of alleles per locus ranged from 2.5 to 3.8 with a mean of 3.0. The genetic variability of this species was comparatively high among aquatic animals. In order to evaluate genetic differences among the nine localities around Japan, the allelic and genotypic homogeneity tests were carried out and Nei\u27s genetic distance was calculated between every pair of the nine localities. Significant differences among localities showed at only a few loci, but genetic distances were low between any pair of localities. Thus, genetic differentiation has not occurred among localities around Japan in A. pectinifera

Estimation of Isozyme Marker Genes and Genetic Variability in Shijimi Clam, Corbicula japonica in Japan

In order to estimate the isozyme marker genes in Shijimi clam, Corbicula japonica, starch gel electrophoresis was carried out about 27 enzymes for three tissues. As a result, twelve isozyme loci, Aat-1, Aat-2, Ak-2, Fdp, Gpi, Idh-1, Mdh-1, Mdh-2, Mdh-3, 6Pgd, Sod-1 and Sod-2 coding for eight enzymes were estimated as genetic markers. Tissue specific patterns were not observed, but foot tissue was better used for isozyme analysis based on clarity of bands. Using the above marker genes, genetic variability was calculated as follows: the proportion of polymorphic loci ranged from 0.17 to 0.42 with a mean of 0.31, expected average heterozygosity was ranged from 0.041 to 0.103 with a mean of 0.071, and mean number of alleles per locus ranged from 1.8 to 2.0 with a mean of 1.9. The results revealed that genetic variability in Corbicula japonica was higher than fishes, crustacean and squids, but lower than the average of marine mollusks. These isozymes could be useful genetic markers for analyzing population structure of this species

The Role of Strategic Bomber as a Means of Transporting Nuclear Weapons

There was an inseparable connection between the development of nuclear weapons and the development of strategic bombers during the period from 1945 to 1968. I discuss the nuclear weapons development competition between the United States of America and the Soviet Union, and clarifies the relationship between nuclear weapons and strategic bombers. And I survey some aircraft accidents that occurred by United States Air Force mission in which B-52 strategic bomber armed with thermonuclear weapons remained on continuous airborne alert, flying routes to points on the Soviet Union border during the period from 1960 to 1968. Finally, I discuss the necessity of disclosing information and removing each other's suspicion

Language and self-name in Northeastern Thailand

The present paper is an attempt to empirically clarify certain aspects of the relationship between language and national or ethnic identity. We use the results of the sociolinguistic survey conducted in Northern Thailand in 2001. Two categories of variables are used: language variables represented by language ability and home language, and identity variables represented by self-name. The results of qualitative correlation/regression analyses in terms of phi coefficients and logistic regression show that; (1) language and self-name of a relative minority positively correlate with each other, as many previous studies argued, (2) a relative majority language, especially its use at home, negatively correlates mutually with the self-name of a relative minority. (3) the self-name of a relative majority is independent of, that is, neither positively nor negatively correlates with, the relative minority language

Microenvironment Modulates Osteogenic Cell Lineage Commitment in Differentiated Embryonic Stem Cells

Background: Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored. Methodology/Principal Findings: ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells. Conclusions/Significance: We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/ differentiation conditions based upon the influence of microenvironment

Young athlete with sudden cardiac arrest treated with therapeutic hypothermia

Reported herein is a coronary anomaly that occurred in a young adolescent athlete who presented with cardiopulmonary arrest. The patient was resuscitated and treated with therapeutic hypothermia. The patient had no associated neurological complications at follow up. Enhanced computed tomography of the heart indicated an anomalous left main coronary artery originating from the right coronary sinus and coursing between the aorta and the pulmonary artery. The patient underwent surgical intervention with coronary artery bypass grafting to prevent symptom recurrence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/100297/1/ped12144.pd

Considerations in hiPSC-derived cartilage for articular cartilage repair

Background: A lack of cell or tissue sources hampers regenerative medicine for articular cartilage damage. Main text: We review and discuss the possible use of pluripotent stem cells as a new source for future clinical use. Human induced pluripotent stem cells (hiPSCs) have several advantages over human embryonic stem cells (hESCs). Methods for the generation of chondrocytes and cartilage from hiPSCs have been developed. To reduce the cost of this regenerative medicine, allogeneic transplantation is preferable. hiPSC-derived cartilage shows low immunogenicity like native cartilage, because the cartilage is avascular and chondrocytes are segregated by the extracellular matrix. In addition, we consider our experience with the aberrant deposition of lipofuscin or melanin on cartilage during the chondrogenic differentiation of hiPSCs. Short conclusion: Cartilage generated from allogeneic hiPSC-derived cartilage can be used to repair articular cartilage damage

Generation of monkey iPS cell-derived cartilage lacking MHC class I molecules on the cell surface

Multiple immune reactions when transplanting cartilage into monkeys. 京都大学プレスリリース. 2021-07-07.Due to the poor capacity for articular cartilage to regenerate, its damage tends to result in progressively degenerating conditions such as osteoarthritis. To repair the damage, the transplantation of allogeneic human induced pluripotent stem cell (iPSC)-derived cartilage is being considered. However, although allogeneic cartilage transplantation is effective, immunological reactions can occur. One hypothetical solution is to delete the expression of MHC class I molecules in order to reduce the immunological reactions. For this purpose, we deleted the β2 microglobulin (B2M) gene in a cynomolgus monkey (crab-eating monkey (Macaca fascicularis)) iPS cells (cyiPSCs) to obtain B2M⁻/⁻ cyiPSCs using the CRISPR/Cas9 system. Western blot analysis confirmed B2M⁻/⁻ cyiPSCs lacked B2M protein, which is necessary for MHC class I molecules to be transported to and expressed on the cell surface by forming multimers with B2M. Flow cytometry analysis revealed no B2M⁻/⁻ cyiPSCs expressed MHC class I molecules on their surface. The transplantation of B2M⁻/⁻ cyiPSCs in immunodeficient mice resulted in teratoma that contained cartilage, indicating that the lack of MHC class I molecules on the cell surface affects neither the pluripotency nor the chondrogenic differentiation capacity of cyiPSCs. By modifying the chondrogenic differentiation protocol for human iPSCs, we succeeded at differentiating B2M⁺/⁺ and B2M⁻/⁻ cyiPSCs toward chondrocytes followed by cartilage formation in vitro, as indicated by histological analysis showing that B2M⁺/⁺ and B2M⁻/⁻ cyiPSC-derived cartilage were positively stained with safranin O and expressed type II collagen. Flow cytometry analysis confirmed that MHC class I molecules were not expressed on the cell surface of B2M⁻/⁻ chondrocytes isolated from B2M⁻/⁻ cyiPSC-derived cartilage. An in vitro mixed lymphocyte reaction assay showed that neither B2M⁺/⁺ nor B2M⁻/⁻ cyiPSC-derived cartilage cells stimulated the proliferation of allogeneic peripheral blood mononuclear cells. On the other hand, osteochondral defects in monkey knee joints that received allogeneic transplantations of cyiPSC-derived cartilage showed an accumulation of leukocytes with more natural killer (NK) cells around B2M⁻/⁻ cyiPSC-derived cartilage than B2M⁺/⁺ cartilage, suggesting complex mechanisms in the immune reaction of allogeneic cartilage transplanted in osteochondral defects in vivo