Biology subjects

Kee, K.

Publications and source records attributed to Kee, K..

4 recordsLinked to original sources

bioRxiv2025

The genomic and transcriptional landscape of the spinal cord H3K27-altered diffuse midline glioma suggests the potential therapeutic strategy

Diffuse midline glioma with H3K27-altered (DMG) is a lethal pediatric malignancy that primarily occurs in the brainstem of children. In adults, DMG arising in the spinal cord is rarely reported and remains poorly understood. The cellular architecture and clinical outcomes of DMG vary depending on the tumors location. To dissect the genomic and transcriptional architecture of a cohort of spinal cord DMG patients, we conducted whole-exome sequencing and single-cell RNA sequencing on 19 tumor samples from 7 patients. We identified previously underappreciated stem-like cells associated with hypoxia in malignant tumor cells, which were present in all samples of spinal cord DMG but absent in published intracranial DMG, corroborating cellular heterogeneity based on tumor location. We discovered that tumor-associated myeloid cells constituted the largest population of non-malignant cells in adult spinal cord DMG. Our research revealed a transition from microglia to macrophages, characterized by enhanced cell-to-cell interactions mediated through the VEGF signaling pathway network. Notably, we found that epigenetic agents can repress the expression of hypoxia-related gene programs in patient-derived spinal cord DMG cultures and inhibit cell proliferation in vitro. Collectively, our study reveals a crucial anatomical dimension that DMG exhibits a location-specific genomic and transcriptional landscape despite shared H3K27 alterations, providing the paradigm for developing precision therapeutic strategies that extend beyond DMG to other molecularly defined yet anatomically divergent malignancies.

cancer biology↗

bioRxiv2022

Single-cell profiling of lncRNAs in human germ cells and molecular analysis reveals transcriptional regulation of LNC1845 on LHX8

Non-coding RNAs exert diverse functions in many cell types. In addition to transcription factors from coding genes, non-coding RNAs may also play essential roles in shaping and directing the fate of germ cells. Here, we report the presence of many long non-coding RNAs (lncRNAs) which were specifically expressed in the germ cells during human gonadal development by single-cell profiling of the reported datasets, and the functional characterization of one divergent lncRNA, LNC1845. Comprehensive bioinformatic analysis of these lncRNAs indicates that divergent lncRNAs occupied the majority of female and male germ cells. Integrating lncRNA expression into the bioinformatic analysis also enhances the cell-type classification of female germ cells. Functional dissection using in vitro differentiation of human pluripotent stem cells to germ cells revealed the regulatory role of LNC1845 on a transcription factor essential for ovarian follicle development, LHX8, by modulating the levels of histone modifications, H3K4me3 and H3K27Ac. Hence, this study provides a comprehensive analysis of lncRNAs in developing germ cells and elucidates how a lncRNA function as a cis regulator during human germ cell development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/486705v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@31290forg.highwire.dtl.DTLVardef@17ec505org.highwire.dtl.DTLVardef@1b8cd0dorg.highwire.dtl.DTLVardef@19e4de3_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIlncRNAs are expressed at significantly higher levels in both male and female germ cells than in somatic cells during human gonadal development. C_LIO_LIDivergent lncRNAs are enriched in the female meiotically and male mitotically arrested germ cells. C_LIO_LILNC1845 regulates the transcriptional expression of LHX8 by modulating H3K4me3 and H3K27Ac levels. C_LI

developmental biology↗

bioRxiv2018

Induction of Sertoli cells from human fibroblasts by NR5A1 and GATA4

Sertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). The induction of human Sertoli cells from fibroblasts could provide cellular sources for fertility and transplantation treatments. Here, we report the in vitro reprogramming of human fibroblasts to Sertoli cells and characterize these human induced Sertoli cells (hiSCs). Initially, five transcriptional factors (NR5A1, GATA4, WT1, SOX9 and DMRT1) and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, i.e., NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles that are similar to those of primary human Sertoli cells. Consistent with the known cellular properties of Sertoli cells, hiSCs attract endothelial cells and exhibit high number of lipid droplets in the cytoplasm. More importantly, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. In addition, hiSCs suppress the production of IL-2 and proliferation of human T lymphocytes. When hiSCs were cotransplanted with human embryonic kidney cells, these xenotransplanted human cells survived longer in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli-only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.

developmental biology↗

bioRxiv2017

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level did not only promote naive pluripotent gene expression but also enhanced cell survival and clonogenicity, and it enabled integration of hESCs with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

cell biology↗