Biology subjects

Otonkoski, T.

Publications and source records attributed to Otonkoski, T..

16 recordsLinked to original sources

bioRxiv2026

Metabolic commitment to human motor neurons is characterized by mitochondrial depletion and pyruvate rewiring

Mitochondrial remodelling is a key feature of neurogenesis in cortical and hippocampal neuronal lineages, yet the temporal control of organellar homeostasis in developing human motor neurons remains poorly understood. Here, we generated a high-resolution temporal resource integrating proteomics, metabolomics, and mitochondrial characterization across human iPSC-to-motor neuron differentiation. This analysis revealed prolonged mitochondrial depletion during early neurogenesis, characterized by reduced mitochondrial content, mitochondrial fragmentation, and lysosome-dependent turnover. This state persisted throughout the neural progenitor stage and was followed by recovery of mitochondrial content during neuronal maturation. During this transition, pyruvate utilization shifted from pyruvate carboxylation towards pyruvate dehydrogenase-dependent oxidation. Pharmacological manipulation of pyruvate partitioning demonstrated that this metabolic switch is required for human neuronal differentiation. Our findings establish a temporal framework for the metabolic control of human motor neuron differentiation and reveal a progenitor-specific state linking mitochondrial depletion to pyruvate metabolism.

cell biology↗

bioRxiv2026

Scinderin-driven Golgi Actin Remodeling coordinates GLP-1 and insulin secretion to regulate glucose homeostasis

Maintenance of glucose homeostasis requires coordinated hormone secretion from intestinal enteroendocrine cells and pancreatic {beta}-cells, yet the intracellular mechanisms that couple nutrient sensing to endocrine output remain poorly defined. Here, we identify the actin remodeler Scinderin (SCIN) as a shared regulator of hormone secretion across these systems. SCIN is selectively expressed in enteroendocrine L-cells and pancreatic {beta}-cells, where it localizes to phosphatidylinositol-4-phosphate (PI(4)P)-enriched Golgi membranes and controls Golgi-associated actin dynamics. Loss of SCIN disrupts Golgi organization, impairs prohormone trafficking, and reduces secretory granule formation, resulting in defective nutrient-stimulated GLP-1 and insulin secretion while preserving cAMP-dependent amplification pathways. In vivo, tissue-specific deletion of Scin compromises incretin responses, {beta}-cell insulin secretion, and systemic glucose homeostasis. Consistent with these findings, SCIN expression is reduced in human diabetic {beta}-cells and associates with stress-related loss of {beta}-cell maturity. Transcriptomic analyses reveal a conserved Golgi stress program upon SCIN loss, linking intracellular trafficking defects to endocrine dysfunction. Together, our results identify SCIN-dependent Golgi actin remodeling as a rate-limiting intracellular mechanism coordinating enteroendocrine and pancreatic hormone secretion. This work uncovers a shared, targetable node controlling endocrine output, providing a mechanistic link between secretory pathway dysfunction and diabetes.

cell biology↗

bioRxiv2026

Loss of mitochondrial co-chaperone GRPEL2 protects mice from age- and diet-induced obesity

Mitochondrial protein homeostasis intersects with metabolic control, but the in vivo roles of specific mitochondrial co-chaperones remain unclear. The chaperone mtHSP70 plays a key role in import and folding of nuclear-encoded proteins targeted to mitochondrial matrix. Its protein folding cycle is regulated by the GrpE-like nucleotide exchange factor GRPEL1. Vertebrates also have a GRPEL2 paralog, postulated as the stress-sensitive counterpart, but its physiological relevance is not known. We show here that GRPEL2 is not essential for viability in mice, and its absence does not induce proteotoxic stress responses in stark contrast to GRPEL1. However, we find that GRPEL2 has a role in regulating body weight homeostasis. GRPEL2 knockout mice are protected from age- and diet-induced weight gain and maintain a better metabolic health and insulin sensitivity. Transcriptional profiling revealed minimal changes in liver and skeletal muscle, whereas white adipose tissue from Grpel2-deficient mice lacked the obesity-associated remodeling seen in controls. We propose that GRPEL2 fine-tunes metabolic setpoints without broadly perturbing mitochondrial protein import, thereby maintaining adipose tissue health during nutritional excess. These findings show that subtle alterations in mitochondrial chaperone systems reshape systemic metabolism and could suggest strategies to mitigate obesity and insulin resistance through targeted modulation of mitochondrial proteostasis.

physiology↗

bioRxiv2025

Ghrelin and relamorelin alleviate hypoglycaemia in humanised mice with congenital hyperinsulinism

KATP-channel related hyperinsulinism (KATPHI) is a genetic disorder of the pancreatic beta cells, manifesting as life-threatening hypoglycaemia in neonates due to dysregulated insulin secretion. Management of the severe diffuse form of KATPHI currently lacks treatment options, as the first-line therapy octreotide is often insufficiently effective, necessitating radical pancreatectomy in many patients. In this study, we applied stem cell derived islets carrying KATPHI-causing mutation KCNJ11-/- to develop new pharmaceutical therapies for KATPHI. We tested seven candidate molecules in vitro, identifying three that reduced insulin secretion in KCNJ11-/- stem cell derived islets. Of these, we tested acyl-ghrelin and its long-acting analogue relamorelin in mice that became hypoglycaemic due to carrying human KCNJ11-/- stem cell derived grafts. Acyl-ghrelin and relamorelin alone increased fasting glycaemia. Combining relamorelin with octreotide increased blood glucose more than the sum of each drug alone, reversing hypoglycaemia to normoglycaemia. In conclusion, we show that ghrelin receptor agonists have acute anti-hypoglycaemic effects in a humanised mouse model of KATPHI, especially when combined with octreotide. Relamorelin has been tested in >650 diabetic adults with little side effects, leading us to propose relamorelin - octreotide combination as a novel therapeutic candidate for severe KATPHI patients.

physiology↗

bioRxiv2024

Effects of alcohol on the transcriptome, methylome, and metabolome of in vitro gastrulating human embryonic cells

Prenatal alcohol exposure (PAE) affects embryonic development, causing a variable fetal alcohol spectrum disorder (FASD) phenotype with neurodevelopmental disorders and birth defects. To explore the effects of PAE on gastrulation, we used an in vitro model with subchronic moderate (20 mM) and severe (70 mM) ethanol exposures during the differentiation of human embryonic stem cells into germ layer cells. We analysed genome-wide gene expression (mRNA sequencing), DNA methylation (EPIC Illumina microarrays), and metabolome (non-targeted LC-MS method) of the endodermal, mesodermal, and ectodermal cells. The largest number of ethanol-induced alterations were observed in the endodermal cells, whereas the most prominent changes were seen in the ectodermal cells. Genes of the major morphogen signaling pathways involved in gastrulation and body patterning were affected by ethanol. Many of the altered genes, such as BMP4, FGF8, SIX3, and LHX2, have been previously associated with PAE and phenotypes of FASD, like defects in heart and corpus callosum development as well as holoprosencephaly. Furthermore, methionine metabolism was altered in all germ layer cells. Our findings support the early origin of alcohol-induced developmental disorders and strengthen the role of methionine cycle in the etiology of FASD.

genetics↗

bioRxiv2023

Quantifying stem cell derived islet graft volume and composition with FDOPA positron emission tomography

Stem cell derived islets (SC-islets) are being developed as a novel source of beta cells that would enable large scale cell replacement therapy for insulin dependent diabetes. Therapeutic use of SC-islets carries an inherent risk of unwanted growth; and multiple strategies are being explored for optimizing long-term SC-islet graft effectiveness. However, a method for noninvasive in vivo monitoring for SC-islet graft safety and efficacy is lacking, as current insulin secretion measurements are inadequate. Here, we demonstrate the potential of positron emission tomography (PET) for monitoring SC-islet grafts using two tracers: GLP1-receptor binding [18F]F-DBCO-exendin and dopamine precursor [18F]FDOPA. We could detect and longitudinally monitor human SC-islet grafts in calf muscles of immunocompromised mice. Importantly, graft volume quantified with PET strongly correlated with actual graft volume (r2=0.91 for [18F]F-DBCO-exendin). PET using [18F]F-DBCO-exendin allowed delineation of cystic structures and its uptake correlated with graft beta cell proportion, enabling study of SC-islet graft purity noninvasively. [18F]FDOPA performed similarly to [18F]F-DBCO-exendin, but with slightly weaker sensitivity. Uptake of neither tracer was biased in SC-islet grafts genetically rendered hyper- or hypoactive. Insulin secretion measurements under fasted, glucose-stimulated or hypoglycemic conditions did not correlate with graft volume. In conclusion, [18F]F-DBCO-exendin and [18F]FDOPA PET constitute powerful approaches to noninvasively assess SC-islet graft volume and composition regardless of their functionality. PET imaging could therefore be leveraged for optimizing safety and effectiveness of SC-islet grafts in patients with insulin dependent diabetes.

cell biology↗

bioRxiv2023

RFX6 haploinsufficiency predisposes to diabetes through impaired beta cell functionality

Regulatory factor X 6 (RFX6) is indispensable for pancreatic endocrine development and differentiation. The RFX6 protein-truncating variant p.His293LeufsTer7 is significantly enriched in the Finnish population with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 diabetes (T2D) and gestational diabetes. To understand the role of this variant in {beta}-cell development and function, we generated allelic series of isogenic pluripotent stem cell models and directed them into pancreatic islet lineages (SC-islets). Expectedly, in-vitro models of the homozygous RFX6-/- variant failed to generate pancreatic endocrine cells, recapitulating the phenotype in Mitchell-Riley syndrome. Notably, heterozygous RFX6+/- derived SC-islets showed reduced {beta}-cell maturation markers and calcium oscillations, resulting in defective insulin secretion, without affecting {beta}-cell number or insulin content. The reduced insulin secretion is sustained during in-vivo implantation studies, consistent with the susceptibility of the carriers to develop diabetes. TeaserModeling RFX6-assocciated neonatal and type-2 diabetes using allelic series stem cell-derived islets in-vitro and in-vivo.

cell biology↗

bioRxiv2023

CRISPR/Cas9 DNA synthesis disruption as a tool to control cell proliferation in vitro and in vivo

Engineered cells hold great promise for regenerative medicine and gene therapy. However, living cell products entail a fundamental biological risk of unwanted growth. Here, we describe a novel metabolic safety system to control cell proliferation without added genetic elements. We inactivated a key enzyme for nucleotide metabolism, TYMS, in several cell lines, thus obtaining cells that proliferate only when supplemented with exogenous thymidine but fail to replicate in its absence. Under supplementation, TYMS-/- pluripotent stem cells proliferate normally, produce teratomas and differentiate into potentially therapeutic cell types such as pancreatic beta cells. After differentiation, the postmitotic cells do not require thymidine to function, as seen by prolonged in vivo production of human insulin in implanted mice. Hence, this method allows robust cell culture and manufacture while mitigating the risk of uncontrolled growth of transplanted cells. One Sentence Summary: Genetic disruption of DNA synthesis prevents unwanted proliferation in cell therapies without affecting cell function.

bioengineering↗

bioRxiv2022

The type 1 diabetes gene TYK2 regulates β-cell development and its responses to interferon-α

Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin producing pancreatic {beta}-cells. One of the genes associated with T1D is TYK2, which encodes a Janus kinase with critical roles in type-I interferon (IFN) mediated intracellular signaling. To study the role of TYK2 in human pancreatic {beta}-cell development and response to IFN, we generated TYK2 knockout human iPSCs and directed them into the pancreatic endocrine lineage. Here we show that loss of TYK2 compromised the emergence of endocrine precursors by regulating KRAS expression while mature stem cell-islets (SC-islets) function was not affected. In the maturing SC-islets, the loss or inhibition of TYK2 prevented IFN-induced antigen processing and presentation, including MHC Class I expression in pancreatic endocrine and progenitor cells. Furthermore, in a CD8+ cytotoxic T-cell co-culture model, the survival of {beta}-cells was enhanced by a selective TYK2 inhibitor. These results identify an unsuspected role for TYK2 on {beta}-cell development and support TYK2 inhibition in adult {beta}-cells as a potent therapeutic target to halt T1D progression.

molecular biology↗

bioRxiv2021

Transient DUX4 expression induces blastomere-like expression program that is marked by SLC34A2

DUX4 has recently been recognized as a key regulator in human embryonic genome activation (EGA). The exact role of DUX4 in human embryo is still elusive, partly due to the cytotoxicity of persistent DUX4 expression in cellular models. We report here that a transient DUX4 expression in human embryonic stem cells (hESCs) retains cell viability while inducing an EGA-like expression program in a subpopulation of the cells. These cells showed resemblance to 8-cell stage blastomeres and were thus named induced blastomere-like (iBM) cells. Trajectory inference from the single-cell RNA-seq data suggested that the expression profile of these cells progressed in a manner similar to the morula to blastocyst transition in human embryo. Finally, viable iBM cells could be enriched using an antibody against NaPi2b (SLC34A2), paving the way for further experimental approaches. The iBM cells can become a powerful tool to model transcriptional dynamics and regulation during early human embryogenesis.

cell biology↗

bioRxiv2021

Functional, metabolic and transcriptional maturation of stem cell derived beta cells

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in stem cell-derived islet (SC-islet) generation, detailed characterization of their functional properties has not been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and comprehensively benchmarked them against primary adult islets. Biphasic glucose stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and increased alpha cells. Electrophysiology and exocytosis of SC-islets were comparable to adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of murine engraftment revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

developmental biology↗

bioRxiv2019

CHCHD10 OR CHCHD2 ARE NOT REQUIRED FOR HUMAN MOTOR NEURON DIFFERENTIATION IN VITRO BUT MODIFY SYNAPTIC TRANSCRIPTOMES

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in diseases affecting motor neurons such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinsons disease, and form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Knockout iPSC and motor neurons sustain mitochondrial ultrastructure and show reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that absence of CHCHD2 or CHCHD10 does not disrupt functionality, but induces similar modifications in human motor neurons. Thus pathogenic mechanisms may involve loss of synaptic function.

neuroscience↗

bioRxiv2019

Maturation of Neuronal Activity in Caudalized Human Brain Organoids

Human brain organoids are an emerging tool to study functional neuronal networks in health and disease. A critical challenge is the engineering of brain organoids with defined regional identity and developmental stage. Here we describe a protocol for generating hindbrain-like organoids from human pluripotent stem cells. We first generated a stable pool of caudalized stem cells that expressed hindbrain identity transcription factors and differentiated into tissue containing neurons and astrocytes. After maturation, caudalized brain organoids presented synaptically connected networks consisting of glutamate-, GABA-, and serotoninergic postmitotic neurons. These mature neurons displayed electric properties and dendritic trees resembling medulla oblongata neurons. They fired spontaneous and evoked repetitive action potentials, released serotonin and displayed excitatory and inhibitory synaptic currents, functionally resembling the activity patterns observed in normal human fetal brain. Reminiscent of infected human fetal brain, infection with Zika virus hampered organoid development, while the treatment with anticonvulsant drugs - carbamazepine and valproic acid - reduced organoid growth. Neuronal maturation also occurred in the grafted organoids in vivo. In conclusion, our approach enables efficient derivation of caudalized neuronal stem cells that differentiate into mature and functional neurons in organoids with hindbrain identity following human developmental trajectory. The organoids provide excellent model to study congenital abnormalities in brain development and for drug testing.

neuroscience↗

bioRxiv2019

DUX4 regulates oocyte to embryo transition in human

During the human oocyte-to-embryo transition, the fertilized oocyte undergoes final maturation and the embryo genome is gradually activated during the first three cell divisions. How this transition is coordinated in humans is largely unknown. We show that the double homeodomain transcription factor DUX4 contributes to this transition. DUX4 knockdown in human zygotes caused insufficient transcriptome reprogramming as observed three days after fertilization. Induced DUX4 expression in human embryonic stem cells activated transcription of thousands of newly identified bi-directional transcripts, including putative enhancers for embryonic genome activation genes such as LEUTX. DUX4 protein interacted with transcriptional modifiers that are known to couple enhancers and promoters. Taken together, our results reveal that DUX4 is a pioneer regulating oocyte-to-embryo transition in human through activation of intergenic genome, especially enhancers, and hence setting the stage for early human embryo development.

developmental biology↗

bioRxiv2018

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion

A rare loss-of-function variant p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8) enriched in Western Finland protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, especially compared with individuals matched for the genotype of a common T2D risk variant in SLC30A8, p.Arg325. In genome-edited human IPS-derived {beta}-like cells, we establish that the p.Arg138* variant results in reduced SLC30A8 expression due to haploinsufficiency. In human {beta}-cells loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function, which was also seen in isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aiming at maintaining insulin secretion capacity in T2D.

genetics↗

bioRxiv2017

Human pluripotent reprogramming with CRISPR activators

CRISPR/Cas9 based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSC) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC and LIN28A promoters. The basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif, enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data constitute a proof of principle that somatic cells can be reprogrammed into iPSC using only CRISPRa. Furthermore, the results unravel previously uncharacterized involvement of EEA-motif-associated mechanisms in cellular reprogramming.

cell biology↗