Biology subjects

Tsakiridis, A.

Publications and source records attributed to Tsakiridis, A..

11 recordsLinked to original sources

bioRxiv2026

Dynamic modelling of human neural crest development using a bioengineered stem cell organoid system

The neural crest (NC) is a transient stem cell population which migrates throughout the developing embryo to contribute to diverse tissues dependent on axial origin. For example, cranial NC can give rise to bone and cartilage, while more posterior NC populations give rise to peripheral nervous system and neuroendocrine tissues. Perturbations in neural crest development can lead to severe congenital anomalies and cancers, with over 700 neurocristopathies reported. In humans, early NC development remains poorly understood due to the inaccessibility of tissue samples, thus necessitating the development of in vitro models. Currently, a limited number of NC organoid protocols are available, but these mainly focus on cranial NC and lack relevant tissue architecture. Here, we describe a novel bioengineered pipeline to derive human pluripotent stem cell (hPSC)-derived neuroepithelial organoids, "neurocrestoids" featuring physiologically-relevant tissue architecture. We show that neurocrestoids recapitulate the dynamics of induction, delamination, and migration of human neural crest cells (NCCs), and can be directly compared to murine NC explants for cross-species validation. Organoids express an array of HOX genes indicating the successful generation of cranial, vagal and trunk NCCs. Moreover, we have integrated our neurocrestoids with a customised micropatterned substrate suitable for live visualisation and guided separation of SOX10-positive migratory human NCCs. Our "NCC migration on-chip" are reproducible across multiple hPSC lines and should be scalable for future diagnostic and therapeutic applications, significantly improving our ability to study human NC pathologies.

developmental biology↗

bioRxiv2026

Notch signalling governs human enteric nervous system progenitor dynamics

The enteric nervous system (ENS) is the main branch of the peripheral nervous system that innervates the gastrointestinal tract controlling vital functions. It arises during embryogenesis via migration, proliferation and differentiation of neural crest-derived ENS progenitors. Perturbation of these processes, caused by mutations in key signalling pathway components and transcription factors, prevents progenitor colonisation of the distal gut causing aganglionic phenotypes and enteric neuropathies such as Hirschsprung (HSCR) disease. While animal models implicate Notch signalling in ENS specification, its role in human ENS progenitor cell fate decisions remains unclear. Here, we employ a human pluripotent stem cell-based model to show that Notch signalling is a key regulator of human ENS progenitor dynamics. Quantitative modelling of our in vitro data indicates that Notch inhibition accelerates progenitor differentiation without substantially altering neuronal versus glial lineage bias. Furthermore, we establish that Notch signalling controls human ENS progenitor migration by influencing migration velocity and directionality. Together, these findings provide mechanistic insights into how Notch signalling disruption may contribute to the pathogenesis of human intestinal aganglionosis.

developmental biology↗

bioRxiv2026

Generation of human hindlimb/genital tubercle progenitors from pluripotent stem cells

The hindlimbs and genital tubercle arise from a bipotent progenitor population within the posterior lateral plate mesoderm (pLPM) and share common genetic programmes, reflecting their deep evolutionary homology. Perturbations of this shared developmental programme underlie several congenital conditions. Yet most insights into the divergence of pLPM fates come from traditional model organisms, emphasising the need for a human-based system. Here, we report the WNT-FGF-BMP-dependent differentiation of human pluripotent stem cells (hPSCs) into pLPM-derived hindlimb/genital tubercle mesenchymal progenitors (HGTps). We show that BMP signalling plays a pivotal role in driving transcriptomic changes reminiscent of the trunk-to-tail transition--a major reorganisation of the embryonic body plan that initiates pLPM/HGTp specification. We further show that retinoic acid signalling exerts a biphasic effect on LPM specification: early exposure blocks trunk-to-tail transition-like transcriptome changes, whereas late exposure enhances genital tubercle mesenchymal fate by suppressing alternative pLPM derivatives. Strikingly, differentiating genital tubercle mesenchyme self-organises with an overlying epithelium resembling in vivo counterparts. Through xenografting approaches, we show that human HGTp-derived spheroids contribute to the genital tubercle region of the chick embryo revealing their developmental potency. Collectively, our work establishes a platform for the reverse engineering and disease modelling of human HGTps.

developmental biology↗

bioRxiv2025

Functional and molecular rescue of aganglionic colon by human enteric nervous system progenitor transplantation in Hirschsprung disease

Hirschsprung disease (HSCR) is a devastating congenital disorder characterised by absence of the enteric nervous system (ENS) in the distal gut. Cell therapy, using human pluripotent stem cell-derived ENS progenitors, offers an attractive alternative to treat this life-limiting disorder. Here we provide an in-depth characterisation of the HSCR phenotype in the B6;129-Ednrbtm1Ywa/J mouse model including molecular analyses highlighting the wide-ranging molecular effects of aganglionosis in HSCR colon. We show that transplantation of hPSC-derived ENS progenitors leads to significant increases in contractile function with formation of extensive donor-derived neuronal networks in colonic explants. Moreover, post-transplantation, we show that integration of ENS progenitors rescues many of the biological pathways impacted in the HSCR aganglionic microenvironment. These novel results provide strong evidence that human ENS progenitor transplantation can modulate critical signalling pathways affected in HSCR and exert positive impacts on contractile function, in aganglionic tissue, supporting further translational development of this regenerative medicine approach.

cell biology↗

bioRxiv2023

Notch signalling influences cell fate decisions and HOX gene induction in axial progenitors

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are critical for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. While the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative feedback loop with FGF signalling.

developmental biology↗

bioRxiv2022

Neuroblastoma-associated chromosomal aberrations drive cell identity loss in human neural crest via disruption of developmental regulators

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

cancer biology↗

bioRxiv2022

HDAC6 inhibition partially alleviates mitochondrial trafficking defects and restores motor function in human motor neuron and zebrafish models of Charcot-Marie-Tooth Disease Type 2A

Charcot Marie Tooth Disease (CMT) is a group of inherited progressive conditions affecting distal motor and sensory neurons, leading to muscle weakness, pain and loss of sensation in limbs. There are currently no treatments for this debilitating disease. To investigate disease mechanisms and facilitate treatment discovery, here we developed an in vitro model for CMT type 2A by introducing the patient-specific MFN2R94Q mutation into human embryonic stem cells (hESCs). Isogenic mutant and wild-type hESCs differentiated to spinal motor neurons with similar efficiency and gave rise to functional motor neurons in vitro. However, MFN2R94Q/+ spinal motor neurons displayed impaired mitochondrial trafficking, resulting in reduced numbers of mitochondria in distal parts of axons. Importantly, we showed that mitochondrial trafficking defects can be alleviated by treatment with an HDAC6 inhibitor. Chemical and genetic inhibition of HDAC6 also significantly rescued the motor phenotype in a zebrafish CMT2A model. Taken together, our study reveals a mutation-specific insight into CMT2A disease mechanism and confirms HDAC6 as a promising target for further therapeutic development.

cell biology↗

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

Defining the signalling determinants of a posterior ventral spinal cord identity in human neuromesodermal progenitor derivatives

The anteroposterior axial identity of motor neurons (MNs) determines their functionality and vulnerability to neurodegeneration. Thus it is a critical parameter in the design of strategies aiming to produce MNs from human pluripotent stem cells (hPSCs) for regenerative medicine and disease modelling applications. However, the in vitro generation of posterior spinal cord MNs has been challenging. Although the induction of cells resembling neuromesodermal progenitors (NMPs), the bona fide precursors of the mammalian spinal cord, offers a promising solution, the progressive specification of posterior MNs from these cells is not well-defined. Here we determine the signals guiding the transition of human NMP-like cells toward posterior ventral spinal cord neurectoderm. We show that combined WNT-FGF activities drive a posterior dorsal early neural state while suppression of TGFβ-BMP signalling pathways, combined with SHH stimulation, promotes a ventral identity. Based on these results, we define an optimised protocol for the generation of posterior MNs that can efficiently integrate within the neural tube of chick embryos. We expect that our findings will facilitate the functional comparison of hPSC-derived spinal cord cells of distinct axial identities.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2019

Retinoic acid accelerates the specification of enteric neural progenitors from in vitro-derived neural crest

The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1-7. Defects in the development and function of the ENS give rise to a range of disorders, termed enteric neuropathies and include conditions such as Hirschsprungs disease. Little is known about the signalling that specifies early ENS progenitors. This has, thus far, limited progress in the generation of enteric neurons from human Pluripotent Stem Cells (hPSCs) that could provide a useful tool for disease modelling and regenerative medicine. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of both vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and following in vivo transplantation, achieving long-term colonisation of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.

developmental biology↗

bioRxiv2018

Human axial progenitors generate trunk neural crest cells

The neural crest (NC) is a multipotent embryonic cell population generating distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and therefore current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

developmental biology↗