Biology subjects

de Lichtenberg, K. H.

Publications and source records attributed to de Lichtenberg, K. H..

6 recordsLinked to original sources

bioRxiv2026

Suspension culture drives metabolic rewiring in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are increasingly expanded in 3D suspension culture to support scalable cell culturing, yet how the transition from 2D to 3D cultures affects hPSCs remains poorly understood. Here, we compare hPSCs in 2D and 3D cultures using transcriptomics, proteomics, spent medium analysis, and stable-isotope tracing. hPSCs maintained pluripotency in both formats, although suspension culture showed altered growth dynamics and increased initial cell loss. Multi-omics analysis identified culture format as the dominant driver of molecular variation and revealed changes in adhesion, cytoskeletal organization, glycolysis, and oxidative phosphorylation. Functional analyses showed increased lactate production from glucose and stable-isotope tracing revealed increased glucose-derived pyruvate formation and altered partitioning of glucose-derived carbon within the TCA cycle in 3D suspension cultures. Glutamine contribution to the TCA cycle was reduced, whereas relative reductive carboxylation was increased in 3D suspension cultures. Together, these findings show that transition from 2D to 3D suspension culture induces metabolic rewiring in hPSCs.

cell biology↗

bioRxiv2025

Efficient Generation of Expandable Dorsal Forebrain Neural Rosette Stem Cell Lines

Neural stem cells (NSCs) represent an interesting option for developing in vitro disease models and drug screening assays due to their differentiation capacity into neurons and glial cells. Additionally, NSCs are under investigation in on-going clinical trials for treatment of various human neurological disorders. NSCs can be isolated from the central nervous system or derived in vitro from human pluripotent stem cells (hPSCs). However, the current methods for generating NSCs typically include a phase of neural rosette formation and subsequent manual isolation of these tiny structures. As this is a laborious process characterized by operator-dependent variability and scalability challenges, there is a pressing need to develop optimized and scalable protocols to obtain pure NSC populations. In this study, we present a new method for generating highly pure and expandable dorsal forebrain FOXG1+OTX2+TLE4+SOX5+ neural rosette stem cell (NRSC) lines without the necessity for manual isolation of rosette structures. Our findings demonstrate the reproducibility of this protocol through the characterization of different NRSC lines over multiple passages, highlighting the robustness of the process. These NRSCs can be expanded for at least 12 passages without compromising their rosette-formation capacity or their initial dorsal forebrain identity. Furthermore, we show the differentiation capacity of these NRSCs to generate pure populations of TUBB3+ neurons, and under specific conditions, their ability to differentiate into early glial progenitor cells including GFAP+ astrocytes and O4+ oligodendrocytes. Collectively, these results show the capabilities of our protocol to generate an expandable NRSC population suitable for in vitro disease modeling and drug screening, while also suggesting a viable strategy for scalable NRSC production for clinical application.

neuroscience↗

bioRxiv2021

TGF-β modulates cell fate in human ES cell-derived foregut endoderm by inhibiting multiple endogenous signaling pathways

Genetic differences between pluripotent stem cell lines causes variable activity of extra-cellular signaling pathways, which limits the reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenously provided factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that TGF-{beta}1 activates an OTX2/LHX1 gene regulatory network that promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, the effects of TGF-{beta}1 cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors make TGF-{beta}1 treated cells refractory to Wnt signaling. Subsequently, TGF-{beta}1-mediated inhibition of Bmp- and Wnt-signaling suppresses liver- and promotes pancreas fate. However, pancreas differentiation is delayed by TGF-{beta}1-induced CYP26A1 expression and inhibition of RA signaling. Our study thus identifies multiple mechanisms of crosstalk between major developmental signaling pathways during foregut patterning.

developmental biology↗

bioRxiv2021

Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network

ObjectiveMice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin secreting beta cells, causing diabetes. In human, homozygous mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function and downstream genetic programs regulated directly by NEUROG3 remain elusive. We therefore mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (iPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. MethodsWe generated a novel hiPSC line (NEUROG3-HA-P2A-Venus), where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs and their NEUROG3-dependence. In addition, we searched whether NEUROG3 binds type 2 diabetes mellitus (T2DM)-associated variants at the PEP stage. ResultsCUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1268 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements frequently overlapping within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of NEUROG3 target genes by FOXA, RFX or PBX transcription factors. Moreover, we found that 22% of the genes downregulated in NEUROG3-/- hESC-derived PEPs are bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 targets include transcription factors known to have important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself. Remarkably, we uncovered that NEUROG3 binds transcriptional regulator genes with enriched expression in human fetal pancreatic alpha (e.g., IRX1, IRX2), beta (e.g., NKX6-1, SMAD9, ISX, TFCP2L1) and delta cells (ERBB4) suggesting that NEUROG3 could control islets subtype programs. Moreover, NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8 and PCSK1). In addition, we unveiled a panel of ncRNA potentially regulated by NEUROG3. Lastly, we identified several T2DM risk SNPs within NEUROG3 peaks suggesting a possible developmental role of NEUROG3 in T2DM susceptibility. ConclusionMapping of NEUROG3 genome occupancy in PEPs uncovers an unexpectedly broad, direct control of the endocrine gene regulatory network (GRN) and raises novel hypotheses on how this master regulator controls islet and beta cell differentiation. HighlightsO_LINEUROG3 CUT&RUN analysis revealed 1268 target genes in human pancreatic endocrine progenitors (PEPs) C_LIO_LINEUROG3 binding sites overlap with active chromatin regions in PEPs. C_LIO_LI1/5 of the genes downregulated in NEUROG3-/- hESC-derived PEPs are bound by NEUROG3. C_LIO_LINEUROG3 targets islet specific TFs and regulators of insulin secretion. C_LIO_LISeveral T2DM risk allelles lie within NEUROG3 bound regions. C_LI

genomics↗

bioRxiv2018

Notch Controls Multiple Pancreatic Cell Fate Regulators Through Direct Hes1-mediated Repression

Notch signaling and its effector Hes1 regulate multiple cell fate choices in the developing pancreas, but few direct target genes are known. Here we use transcriptome analyses combined with chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) to identify direct target genes of Hes1. ChIP-seq analysis of endogenous Hes1 in 266-6 cells, a model of multipotent pancreatic progenitor cells, revealed high-confidence peaks associated with 354 genes. Among these were genes important for tip/trunk segregation such as Ptf1a and Nkx6-1, genes involved in endocrine differentiation such as Insm1 and Dll4, and genes encoding non-pancreatic basic-Helic-Loop-Helix (bHLH) factors such as Neurog2 and Ascl1. Surprisingly, we find that Hes1 binds a large number of loci previously reported to bind Ptf1a, including a site downstream of the Nkx6-1 gene. Notably, we find a number of Hes1 bound genes that are upregulated by {gamma}-secretase inhibition in pancreas explants independently of Neurog3 function, including the tip progenitor/acinar genes; Ptf1a, Gata4, Bhlha15, and Gfi1. Together, our data suggest that Notch signaling suppress the tip cell fate by Hes1-mediated repression of the tip-specific gene regulatory network module that includes transcriptional regulators such as Ptf1a, Gata4, Mist1, and Gfi1. Our data also uncover new molecular targets of Notch signaling that may be important for controlling cell fate choices in pancreas development.

developmental biology↗

bioRxiv2018

Genome-Wide Identification of HES1 Target Genes Uncover Novel Roles for HES1 in Pancreatic Development

Notch signalling and the downstream effector HES1 is required for multiple pancreatic cell fate choices during development, but the direct target genes remain poorly characterised. Here we identify direct HES1 target genes on a genome-wide scale using ChIP-seq and RNA-seq analyses combined with human embryonic stem cell (hESC) directed differentiation of CRISPR/Cas9-generated HES1-/- mutant hESC lines. We found that HES1 binds to a distinct set of endocrine-specific genes, a set of genes encoding basic Helix-Loop-Helix (bHLH) proteins not normally expressed in the pancreas, genes in the Notch pathway, and the known HES1 target NEUROG3. RNA-seq analysis of wild type, HES1-/-, NEUROG3-/-, and HES1-/-NEUROG3-/- mutant hESC lines allowed us to uncover NEUROG3-independent, direct HES1 target genes. Among the HES1 bound genes that were derepressed in HES1-/-NEUROG3-/- cells compared to NEUROG3-/- cells, we found members of the endocrine-specific gene set, the Notch pathway genes DLL1, DLL4, and HEY1, as well as the non-pancreatic bHLH genes ASCL1 and ATOH1. We also found a large number of transcripts specific to the intestinal secretory lineage to be increased in HES1-/-NEUROG3-/- cells. Together, our data reveal that HES1 employs a multi-layered control of endocrine differentiation, controls Notch ligand expression independent of NEUROG3, and prevents initiation of ectopic intestinal transcriptional programmes in pancreas progenitors.

developmental biology↗