Biology subjects

Czerwinski, M.

Publications and source records attributed to Czerwinski, M..

9 recordsLinked to original sources

bioRxiv2025

Expression of a human Gb3/CD77 synthase in insect and human cells: comparison of activity and glycosylation

Glycosylation of proteins can impact their folding, stability, trafficking and enzymatic activity. Human Gb3/CD77 synthase (1,4-galactosyltransferase, A4galt) has two occupied N-glycosylation sites. Previously, we demonstrated that the activity of recombinant enzyme relies on its N-glycosylation. In this study, we produced soluble recombinant catalytic domain of human Gb3/CD77 synthase in two expression hosts known for different glycosylation patterns: Trichoplusia ni insect cells (High Five) and human embryonic kidney cells (Expi293F). The High Five cells generate short oligomannose structures, while the Expi293F cells synthesize complex type glycans. We evaluated the activity of High Five-derived and Expi293F-derived enzymes, characterized the structures of their N-glycans and showed that High Five cells provide a higher amount and activity of the enzyme. Moreover, we used the Expi293F cells to evaluate the N- and C-terminal location of the 6xHis-tag and found that only the N-terminally tagged Expi293F-derived enzyme demonstrated activity. In contrast, the enzyme produced in High Five cells was active despite carrying a C-terminal tag. These findings highlight the role of glycosylation pattern and tag position in the activity of human recombinant glycosyltransferase produced in different hosts. Highlights- Active recombinant Gb3/CD77 synthase was obtained in High Five and Expi293F cells. - High Five cells provided higher recombinant Gb3/CD77 synthase yield than Expi293F. - Glycosylation pattern of recombinant Gb3/CD77 synthase was host specific. - Activity of Expi293F-derived enzyme depended on 6xHis-tag localization.

biochemistry↗

bioRxiv2023

Identification of amnion progenitor-like cells at the amnion-epiblast boundary in the primate peri-gastrula

Amniogenesis is triggered in a collection of pluripotent epiblast cells as the human embryo implants. To gain insights into the critical but poorly understood transcriptional machinery governing amnion fate determination, we examined the evolving transcriptome of a developing human pluripotent stem cell-derived amnion model at the single cell level. This analysis revealed several continuous amniotic fate progressing states with state-specific markers, which include a previously unrecognized CLDN10+ amnion progenitor state. Strikingly, we found that expression of CLDN10 is restricted to the amnion-epiblast boundary region in the human post-implantation amniotic sac model as well as in a peri-gastrula cynomolgus macaque embryo, bolstering the growing notion that, at this stage, the amnion-epiblast boundary is a site of active amniogenesis. Bioinformatic analysis of published primate peri-gastrula single cell sequencing data further confirmed that CLDN10 is expressed in cells progressing to amnion. Additionally, our loss of function analysis shows that CLDN10 promotes amniotic but suppresses primordial germ cell-like fate. Overall, this study presents a comprehensive amniogenic single cell transcriptomic resource and identifies a previously unrecognized CLDN10+ amnion progenitor population at the amnion-epiblast boundary of the primate peri-gastrula.

developmental biology↗

bioRxiv2021

Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase curtails its activity

N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Gal1[->]4Gal (Gb3 and the P1 antigen) and Gal1[->]4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins.

biochemistry↗

bioRxiv2020

What we can and what we cannot see with extracellular multielectrodes

Extracellular recording is an accessible technique used in animals and humans to study the brain physiology and pathology. As the number of recording channels and their density grows it is natural to ask how much improvement the additional channels bring in and how we can optimally use the new capabilities for monitoring the brain. Here we show that for any given distribution of electrodes we can establish exactly what information about current sources in the brain can be recovered and what information is strictly unobservable. We demonstrate this in the general setting of previously proposed kernel Current Source Density method and illustrate it with simplified examples as well as using evoked potentials from the barrel cortex obtained with a Neuropixels probe and with compatible model data. Author SummaryEvery set of measurements is a window into reality rendering its incomplete or distorted picture. It is often difficult to relate the obtained representation of the world to underlying ground truth. Here we show, for brain electrophysiology, for arbitrary experimental setup (distribution of electrodes), and arbitrary analytical setup (function space of current source densities), that one can identify distributions of current sources which can be recovered precisely, and those which are invisible in the system. This shows what is and what is not observable in the studied system for a given setup, allows to improve the analysis results by modifying analytical setup, and facilitates interpretation of the measured sets of LFP, ECoG and EEG recordings.

neuroscience↗

bioRxiv2020

An organoid and multi-organ developmental cell atlas reveals multilineage fate specification in the human intestine

Human intestinal organoids (HIOs) generated from pluripotent stem cells provide extraordinary opportunities to explore development and disease. Here, we generate a single-cell transcriptome reference atlas from HIOs and from multiple developing human organs to quantify the specificity of HIO cell fate acquisition, and to explore alternative fates. We identify epithelium-mesenchyme interactions, transcriptional regulators involved in cell fate specification, and stem cell maturation features in the primary tissue that are recapitulated in HIOs. We use an HIO time course to reconstruct the molecular dynamics of intestinal stem cell emergence, as well as the specification of multiple mesenchyme subtypes. We find that the intestinal master regulator CDX2 correlates with distinct phases of epithelial and mesenchymal development, and CDX2 deletion perturbs the differentiation of both intestinal epithelium and mesenchyme. Collectively our data provides a comprehensive and quantitative assessment of HIO development, and illuminates the molecular machinery underlying endodermal and mesodermal cell fate specification.

developmental biology↗

bioRxiv2020

Differentiation of human intestinal organoids with endogenous vascular endothelial cells

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) generated using directed differentiation lack some cellular populations found in the native organ, including vasculature. Using single cell RNA sequencing (scRNAseq), we have identified a transient population of endothelial cells (ECs) present early in HIO differentiation that are lost over time in culture. Here, we have developed a method to enhance co-differentiation and maintenance of ECs within HIOs (vHIOs). Given that ECs are known to possess organ specific gene expression, morphology and function, we used bulk RNAseq and scRNAseq to interrogate the developing human intestine, lung, and kidney in order to identify organ-enriched EC-gene signatures in these organ systems. By comparing organ-specific gene signatures along with markers validated by fluorescent in situ hybridization to HIO ECs, we find that HIO ECs grown in vitro share the highest similarity with native intestinal ECs relative to kidney and lung. Together, these data show that HIOs can co-differentiate a native EC population that are properly patterned with an intestine-specific EC transcriptional signature in vitro.

developmental biology↗

bioRxiv2020

In vitro and in vivo development of the human intestine at single cell resolution

The human intestinal stem cell (ISC) niche supports ISC self-renewal and epithelial function, yet little is known about the development of the human ISC niche. We used single-cell mRNA sequencing (scRNA-seq) to interrogate the human intestine across 7-21 weeks of gestation. Using these data coupled with marker validation in situ, molecular identities and spatial locations were assigned to several cell populations that comprise the epithelial niche, and the cellular origins of many niche factors were determined. The major source of WNT and RSPONDIN ligands were ACTA2+ cells of the muscularis mucosa. EGF was predominantly expressed in the villus epithelium and the EGF-family member NEUREGULIN1 (NRG1) was expressed by subepithelial mesenchymal cells. Functional data from enteroid cultures showed that NRG1 improved cellular diversity, enhanced the stem cell gene signature, and increased enteroid forming efficiency, whereas EGF supported a secretory gene expression profile and stimulated rapid proliferation. This work highlights unappreciated complexities of intestinal EGF/ERBB signaling and identifies NRG1 as a stem cell niche factor.

developmental biology↗

bioRxiv2018

Basal stem cell fate specification is mediated by SMAD signaling in the developing human lung

Basal stem cells (basal cells), located in the bronchi and trachea of the human lung epithelium, play a critical role in normal airway homeostasis and repair, and have been implicated in the development of diseases such as cancer1-4. Additionally, basal-like cells contribute to alveolar regeneration and fibrosis following severe injury5-8. However, the developmental origin of basal cells in humans is unclear. Previous work has shown that specialized progenitor cells exist at the tips of epithelial tubes during lung branching morphogenesis, and in mice, give rise to all alveolar and airway lineages9,10. These bud tip progenitor cells have also been described in the developing human lung11-13, but the mechanisms controlling bud tip differentiation into specific cell lineages, including basal cells, are unknown. Here, we interrogated the bud tip-to-basal cell transition using human tissue specimens, bud tip progenitor organoid cultures11, and single-cell transcriptomics. We used single-cell mRNA sequencing (scRNAseq) of developing human lung specimens from 15-21 weeks gestation to identify molecular signatures and cell states in the developing human airway epithelium. We then inferred differentiation trajectories during bud tip-to-airway differentiation, which revealed a previously undescribed transitional cell state ( hub progenitors) and implicated SMAD signaling as a regulator of the bud tip-to-basal cell transition. We used bud tip progenitor organoids to show that TGFT1 and BMP4 mediated SMAD signaling robustly induced the transition into functional basal-like cells, and these in vitro-derived basal cells exhibited clonal expansion, self-renewal and multilineage differentiation. This work provides a framework for deducing and validating key regulators of cell fate decisions using single cell transcriptomics and human organoid models. Further, the identification of SMAD signaling as a critical regulator of newly born basal cells in the lung may have implications for regenerative medicine, basal cell development in other organs, and understanding basal cell misregulation in disease.

developmental biology↗

bioRxiv2018

Non-adhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids

Human intestinal organoids (HIOs) represent a powerful system to study human development and are promising candidates for clinical translation as drug-screening tools or engineered tissue. Experimental control and clinical use of HIOs is limited by growth in expensive and poorly defined tumor-cell-derived extracellular matrices, prompting investigation of synthetic ECM-mimetics for HIO culture. Since HIOs possess an inner epithelium and outer mesenchyme, we hypothesized that adhesive cues provided by the matrix may be dispensable for HIO culture. Here, we demonstrate that alginate, a minimally supportive hydrogel with no inherent cell adhesion properties, supports HIO growth in vitro and leads to HIO epithelial differentiation that is virtually indistinguishable from Matrigel-grown HIOs. Additionally, alginate-grown HIOs mature to a similar degree as Matrigel-grown HIOs when transplanted in vivo, both resembling human fetal intestine. This work demonstrates that purely mechanical support from a simple-to-use and inexpensive hydrogel is sufficient to promote HIO survival and development.

developmental biology↗