Biology subjects

Takasato, M.

Publications and source records attributed to Takasato, M..

7 recordsLinked to original sources

bioRxiv2026

Time-dependent BMP4 signaling directs lineage specification in human mesoderm

Human pluripotent stem cells (hPSCs) provide a powerful platform for modeling early human embryonic development. Here, we investigate the mechanisms underlying mesodermal heterogeneity using a minimal directed differentiation system that simultaneously generates paraxial (PXM), intermediate (IM) and lateral plate mesoderm (LPM) populations. Single-cell RNA sequencing across defined time points during hPSC differentiation revealed a temporal sequence of lineage specification with LPM emerging first, followed by PXM and IM differentiation. Ligand-receptor and differential gene expression analyses identified BMP4 as a key regulator enriched in LPM-associated clusters versus mesoderm progenitors (MPs) that hold PXM and IM precursors. Whereas LPM cells cluster with an early BMP4 signal, IM clusters are associated with later BMP4. Moreover, these early and late BMP4 signals regulate this lineage specification potentially through distinct downstream pathways. Leveraging this insight, we established a stepwise protocol combining early BMP inhibition with subsequent BMP4 supplementation, suppressing initial LPM fate to efficiently induce IM from a mixed MP population. Longer culture of these selective IM progenitors promotes more mature nephrogenesis. Moreover, we demonstrate that during early differentiation high levels of BMP4 can still redirect MPs to more lateroventral fates, illustrating a degree of plasticity within the mesoderm lineage. Together, our results define a temporal framework for BMP4 signaling in mesoderm fate determination and provide a strategy for selective mesoderm differentiation from hPSCs. HIGHLIGHTSO_LIDevelopment of a minimal 2D differentiation platform allows for heterogenous mesoderm formation. C_LIO_LITemporal BMP4 signaling differentially directs mesoderm fates, with early exposure favoring LPM and late exposure promoting IM identity. C_LIO_LILPM cells arise first while later mesoderm progenitors hold both IM and PXM-fated cells. C_LIO_LISequential BMP modulation promotes IM and enhances nephrogenesis. C_LI

developmental biology↗

bioRxiv2023

Identification of three distinct cell populations for urate excretion in human kidney

In humans, uric acid is an end-product of purine metabolism. Urate excretion from human kidney is tightly regulated by reabsorption and secretion. At least eleven genes have been identified as human renal urate transporters. However, it remains unclear whether all renal tubular cells express the same set of urate transporters. Here we show that renal tubular cells are divided into three distinct cell populations for urate handling. Analysis of healthy human kidneys at single-cell resolution revealed that not all renal tubular cells expressed the same set of urate transporters. Only 32% of renal tubular cells were related to both reabsorption and secretion, while the remaining renal tubular cells were related to either reabsorption or secretion, at 5% and 63% respectively. These results provide physiological insight into the molecular function of the transporters and renal urate handling on cell-units. Our findings also suggest that three different tubular cell populations cooperate to regulate urate excretion from human kidney. Highlight/Key pointsO_LIWe identified three distinct cell populations within the human renal anatomy that predict putative cellular transport mode, and our findings indicate cellular inhomogeneity with distinct roles such as urate secretion and reabsorption. C_LIO_LIOur model of physiological urate handling demonstrates the excretion dynamics in human kidney in terms of single cell-units. C_LIO_LIOur cellular urate transport analyses suggest the reversibility of some urate transporters even in certain physiological conditions. C_LIO_LIThe physiological function of SLC2A9 is not limited to urate reabsorption; it is also involved in urate secretion restriction. C_LIO_LIThis methodology can be applied to investigations of transport mechanisms in general, regardless of epithelial cell types, species, and substrates. C_LI

bioinformatics↗

bioRxiv2021

Early anteroposterior regionalisation of human neural crest is shaped by a pro-mesodermal factor

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cells, the presumed precursors of the childhood tumour neuroblastoma, are derived from neuromesodermal-potent progenitors of the postcranial body (NMPs). Here we employ human embryonic stem cell differentiation to define how NMP-derived NC cells acquire a posterior axial identity. We show that TBXT, a pro-mesodermal transcription factor, mediates early posterior NC regionalisation together with WNT signalling effectors. This occurs by TBXT-driven chromatin remodelling via its binding in key enhancers within HOX gene clusters and other posterior regulator-associated loci. In contrast, posteriorisation of NMP-derived spinal cord cells is TBXT/WNT-independent and takes place under the influence of FGF signalling. Our work reveals a previously unknown role of TBXT in influencing posterior NC fate and points to the existence of temporally discrete, cell type-dependent modes of posterior axial identity control.

developmental biology↗

bioRxiv2020

Reprogramming Epiblast Stem Cells into Pre-Implantation Blastocyst Cell-like Cells

Recently, a new wave of synthetic embryo systems (SESs) have been established from cultured cells toward efficient and ethical embryonic development research. We recently reported our epiblast stem cell (EPISC) reprogramming SES that generates numerous blastocyst (BC)-like hemispheres (BCLH) with pluripotent and extraembryonic cell features detected microscopically. Here, we further explored the system over key time points with unprecedented single-cell RNA sequencing (scRNA-seq) analysis and revealed broad induction of the 2C-like reporter MERVL and RNA velocity diverging three major population regions with genetic expression resembling pluripotent epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). Enrichment of those three BC-like cell fates involved key regulons, zygotic genome activation (ZGA) related genes, specific RNA splicing, and select cells meaningfully distinguished critical regulons of model cells. This analysis confirms the induction of the extraembryonic cell populations during the reprogramming and we anticipate that our unique BCLH SES and rich data may uncover new facets of cell potency, improve developmental biology, and help biomedicine advance.

synthetic biology↗

bioRxiv2020

Inducing Human Retinal Pigment Epithelium-like Cells from Somatic Tissue

Regenerative medicine relies on basic research to find safe and useful outcomes that are only practical when cost-effective. The human eyeball requires the retinal pigment epithelium (RPE) for support and maintenance that interfaces the neural retina and the choroid at large. Nearly 200 million people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among other retinal pathologies related to RPE degradation. Recently, autologous pluripotent stem cell-derived RPE cells were prohibitively expensive due to time, therefore we developed a new simplified cell reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts by conditional overexpression of broad plasticity and lineage-specific pioneering transcription factors. iRPE cells showed features of modern RPE benchmarks and significant in-vivo integration in transplanted chimeric hosts. Herein, we detail the iRPE system with comprehensive modern single-cell RNA (scRNA) sequencing profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for regenerative medicine research and affordable autologous human RPE tissue for cell therapy.

bioengineering↗

bioRxiv2019

Human URAT1/SLC22A12 gene promoter is regulated by 27-hydroxycholesterol through estrogen response elements

Elevated levels of uric acid, a metabolite of purine in humans, is related to various diseases, such as gout, atherosclerosis and renal dysfunction. The excretion and reabsorption of uric acid to/from urine is tightly regulated by uric acid transporters. The amino acid sequences of uric acid reabsorption transporters, URAT1/SLC22A12, OAT4/SLC22A11, and OAT10/SLC22A13, share closer phylogenic relationship, whereas the gene promoter sequences are distant phylogenic relationship. Through the single-cell RNA-sequencing analysis of an adult human kidney, we found that only a small number of cells express these transporters, despite their role in the regulation of serum uric acid levels. Transcriptional motif analysis on these transporter genes, revealed that the URAT1/SLC22A12 gene promoter displayed the most conserved estrogen response elements (EREs) among the three transporters. The endogenous selective estrogen receptor modulator (SERM) 27-hydroxycholesterol (27HC) had positive effects on the transcriptional activity of URAT1/SLC22A12. We also found that 27HC increased the protein and gene expression of URAT1/SLC22A12 in mouse kidneys and human kidney organoids, respectively. These results strongly suggest the role of 27HC for URAT1/SLC22A12 expression in renal proximal tubules and upregulation of serum uric acid levels and also show the relationship between cholesterol metabolism and serum uric acid regulation. Significance StatementThe elevated levels of serum uric acid cause various diseases, and the excretion/reabsorption of uric acid to/from urine is tightly regulated by the uric acid transporters. We found that despite the role in serum uric acid regulation, only a small number of cells express URAT1/SLC22A12. We also found that URAT1/SLC22A12 gene promoter region has effective estrogen response elements, and endogenous selective estrogen receptor (ER) modulator 27-hydroxycholesterol (27HC) increased URAT1/SLC22A12 expression in the mice kidneys and human kidney organoids. These suggest that 27HC increases URAT1/SLC22A12 expression and upregulate serum uric acid levels. Since 27HC connects cholesterol metabolism, our study indicates the important link between cholesterol metabolism and serum uric acid regulation, and also provides a novel therapeutic approach to hyperuricemia.

biochemistry↗

bioRxiv2017

Transcriptional evaluation of the developmental accuracy, reproducibility and robustness of kidney organoids derived from human pluripotent stem cells

We have previously reported a protocol for the directed differentiation of human induced pluripotent stem cells to kidney organoids comprised of nephrons, proximal and distal epithelium, vasculature and surrounding interstitial elements. The utility of this protocol for applications such as disease modelling will rely implicitly on the developmental accuracy of the model, technical robustness of the protocol and transferability between iPSC lines. Here we report extensive transcriptional analyses of the sources of variation across the timecourse of differentiation from pluripotency to complete kidney organoid, focussing on repeated differentiations to day 18 organoid. Individual organoids generated within the same differentiation experiment show Spearmans correlation coefficients of >0.99. The greatest source of variation was seen between experimental batch, with the enrichment for genes that also varied temporally between day 10 and day 25 organoids implicating nephron maturation as contributing to transcriptional variance between individual differentiation experiments. A morphological analysis revealed a transition from renal vesicle to capillary loop stage nephrons across the same time period. Distinct iPSC clones were also shown to display congruent transcriptional programs with inter-experimental and inter-clonal variation most strongly associated with nephron patterning. Even epithelial cells isolated from organoids showed transcriptional alignment with total organoids of the same day of differentiation. This data provides a framework for managing experimental variation, thereby increasing the utility of this approach for personalised medicine and functional genomics.

bioinformatics↗