Modeling human somite development and fibrodysplasia ossificans progressiva with induced pluripotent stem cells.

Somites (SMs) comprise a transient stem cell population that gives rise to multiple cell types, including dermatome (D), myotome (MYO), sclerotome (SCL) and syndetome (SYN) cells. Although several groups have reported induction protocols for MYO and SCL from pluripotent stem cells, no studies have demonstrated the induction of SYN and D from SMs. Here, we report systematic induction of these cells from human induced pluripotent stem cells (iPSCs) under chemically defined conditions. We also successfully induced cells with differentiation capacities similar to those of multipotent mesenchymal stromal cells (MSC-like cells) from SMs. To evaluate the usefulness of these protocols, we conducted disease modeling of fibrodysplasia ossificans progressiva (FOP), an inherited disease that is characterized by heterotopic endochondral ossification in soft tissues after birth. Importantly, FOP-iPSC-derived MSC-like cells showed enhanced chondrogenesis, whereas FOP-iPSC-derived SCL did not, possibly recapitulating normal embryonic skeletogenesis in FOP and cell-type specificity of FOP phenotypes. These results demonstrate the usefulness of multipotent SMs for disease modeling and future cell-based therapies.

Development of pluripotent stem cell-based human tenocytes.

Human pluripotent stem cells (PSCs) are used as a platform for therapeutic purposes such as cell transplantation therapy and drug discovery. Another motivation for studying PSCs is to understand human embryogenesis and development. All cell types that make up the body tissues develop through defined trajectories during embryogenesis. For example, paraxial mesoderm is considered to differentiate into several cell types including skeletal muscle cells, chondrocytes, osteocytes, dermal fibroblasts, and tenocytes. Tenocytes are fibroblast cells that constitute the tendon. The step-wise narrowing fate decisions of paraxial mesoderm in the embryo have been modeled in vitro using PSCs; however, deriving tenocytes from human-induced PSCs and their application in cell therapy have long been challenging. PSC-derived tenocytes can be used for a source of cell transplantation to treat a damaged or ruptured tendon due to injury, disorder, or aging. In this review, we discuss the latest research findings on the use of PSCs for studying the biology of tenocyte development and their application in therapeutic settings.

Transglutaminase mediates the hardening of fish egg envelope produced by duplication of factor XIIIA gene during the evolution of Teleostei.

During the fertilization of fish eggs, the hardening of the egg envelope is mediated by transglutaminase (hTGase). After fertilization, TGase undergoes processing. We isolated hTGase from extracts of unfertilized and water-activated rainbow trout eggs. Rainbow trout hTGase (Rt-hTGase) appeared as an 80 kDa protein, and its processed form was 55 kDa. Their N-terminal amino acid sequences were nearly identical, suggesting processing in the C-terminal region. The specific activities were not significantly different, indicating that C-terminal processing does not activate the enzyme itself. We cloned the cDNA by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers followed by RACE-PCR. The deduced amino acid sequence of the cDNA was similar to that of factor XIII subunit A (FXIIIA). Molecular phylogenetic and gene syntenic analyses clearly showed that hTGase was produced by duplication of FXIIIA during the evolution to Teleostei. The 55 kDa processed form of Rt-hTGase is predominantly composed of an enzyme domain predicted from the amino acid sequence of the cDNA. It is hypothesized that the C-terminal domain of Rt-hTGase binds to egg envelope proteins, and that processing allows the enzyme to move freely within the egg envelope, increasing substrate-enzyme interaction and thereby accelerating hardening.

Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture.

Tendon self-renewal is a rare occurrence because of the poor vascularization of this tissue; therefore, reconstructive surgery using autologous tendon is often performed in severe injury cases. However, the post-surgery re-injury rate is relatively high, and the collection of autologous tendons leads to muscle weakness, resulting in prolonged rehabilitation. Here, we introduce an induced pluripotent stem cell (iPSC)-based technology to develop a therapeutic option for tendon injury. First, we derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in vertebrate embryos. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSC-derived tenocytes. We demonstrated that iPSC-tenocyte grafting contributed to motor function recovery after Achilles tendon injury in rats via engraftment and paracrine effects. The biomechanical strength of regenerated tendons was comparable to that of healthy tendons. We suggest that iPSC-tenocytes will provide a therapeutic option for tendon injury.

A Modular Differentiation System Maps Multiple Human Kidney Lineages from Pluripotent Stem Cells.

Recent studies using human pluripotent stem cells (hPSCs) have developed protocols to induce kidney-lineage cells and reconstruct kidney organoids. However, the separate generation of metanephric nephron progenitors (NPs), mesonephric NPs, and ureteric bud (UB) cells, which constitute embryonic kidneys, in in vitro differentiation culture systems has not been fully investigated. Here, we create a culture system in which these mesoderm-like cell types and paraxial and lateral plate mesoderm-like cells are separately generated from hPSCs. We recapitulate nephrogenic niches from separately induced metanephric NP-like and UB-like cells, which are subsequently differentiated into glomeruli, renal tubules, and collecting ducts in vitro and further vascularized in vivo. Our selective differentiation protocols should contribute to understanding the mechanisms underlying human kidney development and disease and also supply cell sources for regenerative therapies.

Insights into the biology of fibrodysplasia ossificans progressiva using patient-derived induced pluripotent stem cells.

The demand for development of new drugs remains on the upward trend because of the large number of patients suffering from intractable diseases for which effective treatment has not been established yet. Recently, several researchers have attempted to apply induced pluripotent stem cell (iPSC) technology as a powerful tool for studying the mechanisms underlying the onset of various diseases and for new drug screening. This technology has made an enormous breakthrough, since it permits us to recapitulate the disease phenotype in vitro, outside of the patient's body. Here, we discuss the latest findings that uncovered a mechanism underlying the pathology of a rare genetic musculoskeletal disease, fibrodysplasia ossificans progressiva (FOP), by modeling the phenotypes with FOP patient-derived iPSCs, and that discovered promising candidate drugs for FOP treatment. We also discussed future directions of FOP research.

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

Differential microarray analysis of Drosophila mushroom body transcripts using chemical ablation.

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain. As a way to systematically elucidate genes preferentially expressed in MBs, we have analyzed genome-wide alterations in transcript profiles associated with MB ablation by hydroxyurea. We selected 100 genes based on microarray data and examined their expression patterns in the brain by in situ hybridization. Seventy genes were found to be expressed in the posterodorsal cortex, which harbors the MB cell bodies. These genes encode proteins of diverse functions, including transcription, signaling, cell adhesion, channels, and transporters. Moreover, we have examined developmental functions of 40 of the microarray-identified genes by transgenic RNA interference; 8 genes were found to cause mild-to-strong MB defects when suppressed with a MB-Gal4 driver. These results provide important information not only on the repertoire of genes that control MB development but also on the repertoire of neural factors that may have important physiological functions in MB plasticity.