-
The Journal of Clinical Investigation Mar 2024Allergic asthma generally starts during early life and is linked to substantial tissue remodeling and lung dysfunction. Although angiogenesis is a feature of the...
Allergic asthma generally starts during early life and is linked to substantial tissue remodeling and lung dysfunction. Although angiogenesis is a feature of the disrupted airway, the impact of allergic asthma on the pulmonary microcirculation during early life is unknown. Here, using quantitative imaging in precision-cut lung slices (PCLSs), we report that exposure of neonatal mice to house dust mite (HDM) extract disrupts endothelial cell/pericyte interactions in adventitial areas. Central to the blood vessel structure, the loss of pericyte coverage was driven by mast cell (MC) proteases, such as tryptase, that can induce pericyte retraction and loss of the critical adhesion molecule N-cadherin. Furthermore, spatial transcriptomics of pediatric asthmatic endobronchial biopsies suggests intense vascular stress and remodeling linked with increased expression of MC activation pathways in regions enriched in blood vessels. These data provide previously unappreciated insights into the pathophysiology of allergic asthma with potential long-term vascular defects.
Topics: Humans; Child; Animals; Mice; Mast Cells; Pericytes; Endothelial Cells; Asthma; Lung; Allergens; Pyroglyphidae; Disease Models, Animal
PubMed: 38487999
DOI: 10.1172/JCI173676 -
Cytotherapy Jun 2024Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we...
BACKGROUND AIMS
Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products.
METHODS
Our standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs).
RESULTS
Two MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm).
CONCLUSIONS
We successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.
Topics: Humans; Induced Pluripotent Stem Cells; Cell Differentiation; Cell Culture Techniques; Cell Line
PubMed: 38483359
DOI: 10.1016/j.jcyt.2024.02.021 -
Alzheimer's Research & Therapy Mar 2024Although abnormal accumulation of amyloid beta (Aβ) protein is thought to be the main cause of Alzheimer's disease (AD), emerging evidence suggests a pivotal vascular...
BACKGROUND
Although abnormal accumulation of amyloid beta (Aβ) protein is thought to be the main cause of Alzheimer's disease (AD), emerging evidence suggests a pivotal vascular contribution to AD. Aberrant amyloid β induces neurovascular dysfunction, leading to changes in the morphology and function of the microvasculature. However, little is known about the underlying mechanisms between Aβ deposition and vascular injuries. Recent studies have revealed that pericytes play a substantial role in the vasculopathy of AD. Additional research is imperative to attain a more comprehensive understanding.
METHODS
Two-photon microscopy and laser speckle imaging were used to examine cerebrovascular dysfunction. Aβ oligomer stereotactic injection model was established to explain the relationship between Aβ and vasculopathy. Immunofluorescence staining, western blot, and real-time PCR were applied to detect the morphological and molecular alternations of pericytes. Primary cultured pericytes and bEnd.3 cells were employed to explore the underlying mechanisms.
RESULTS
Vasculopathy including BBB damage, hypoperfusion, and low vessel density were found in the cortex of 8 to 10-month-old 5xFAD mice. A similar phenomenon accompanied by pericyte degeneration appeared in an Aβ-injected model, suggesting a direct relationship between Aβ and vascular dysfunction. Pericytes showed impaired features including low PDGFRβ expression and increased pro-inflammatory chemokines secretion under the administration of Aβ in vitro, of which supernatant cultured with bEND.3 cells led to significant endothelial dysfunction characterized by TJ protein deficiency.
CONCLUSIONS
Our results provide new insights into the pathogenic mechanism underlying Aβ-induced vasculopathy. Targeting pericyte therapies are promising to ameliorate vascular dysfunction in AD.
Topics: Mice; Animals; Amyloid beta-Peptides; Pericytes; Blood-Brain Barrier; Endothelial Cells; Alzheimer Disease; Cerebrovascular Disorders; Cerebral Amyloid Angiopathy
PubMed: 38475929
DOI: 10.1186/s13195-024-01423-w -
BioRxiv : the Preprint Server For... Feb 2024The primitive gut tube of mammals initially forms as a simple cylinder consisting of the endoderm-derived, pseudostratified epithelium and the mesoderm-derived...
The primitive gut tube of mammals initially forms as a simple cylinder consisting of the endoderm-derived, pseudostratified epithelium and the mesoderm-derived surrounding mesenchyme. During mid-gestation a dramatic transformation occurs in which the epithelium is both restructured into its final cuboidal form and simultaneously folded and refolded to create intestinal villi and intervillus regions, the incipient crypts. Here we show that the mesenchymal winged helix transcription factor Foxl1, itself induced by epithelial hedgehog signaling, controls villification by activating BMP and PDGFRα as well as planar cell polarity genes in epithelial-adjacent telocyte progenitors, both directly and in a feed-forward loop with Foxo3.
PubMed: 38464137
DOI: 10.1101/2024.02.27.582300 -
The Journal of Biological Chemistry May 2024Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) oncogenic fusion proteins are found in approximately 5% of non-small cell lung...
Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) oncogenic fusion proteins are found in approximately 5% of non-small cell lung cancers. Different EML4-ALK fusion variants exist with variant 3 (V3) being associated with a significantly higher risk than other common variants, such as variant 1 (V1). Patients with V3 respond less well to targeted ALK inhibitors, have accelerated rates of metastasis, and have poorer overall survival. A pathway has been described downstream of EML4-ALK V3 that is independent of ALK catalytic activity but dependent on the NEK9 and NEK7 kinases. It has been proposed that assembly of an EML4-ALK V3-NEK9-NEK7 complex on microtubules leads to cells developing a mesenchymal-like morphology and exhibiting enhanced migration. However, downstream targets of this complex remain unknown. Here, we show that the microtubule-based kinesin, Eg5, is recruited to interphase microtubules in cells expressing EML4-ALK V3, whereas chemical inhibition of Eg5 reverses the mesenchymal morphology of cells. Furthermore, we show that depletion of NEK7 interferes with Eg5 recruitment to microtubules in cells expressing EML4-ALK V3 and cell length is reduced, but this is reversed by coexpression of a phosphomimetic mutant of Eg5, in a site, S1033, phosphorylated by NEK7. Intriguingly, we also found that expression of Eg5-S1033D led to cells expressing EML4-ALK V1 adopting a more mesenchymal-like morphology. Together, we propose that Eg5 acts as a substrate of NEK7 in cells expressing EML4-ALK V3 and Eg5 phosphorylation promotes the mesenchymal morphology typical of these cells.
Topics: NIMA-Related Kinases; Humans; Phosphorylation; Oncogene Proteins, Fusion; Kinesins; Microtubules; Lung Neoplasms; Carcinoma, Non-Small-Cell Lung; Mesoderm; Cell Line, Tumor; Protein Serine-Threonine Kinases
PubMed: 38458397
DOI: 10.1016/j.jbc.2024.107144 -
Stem Cell Research Apr 2024The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital...
The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease.
Topics: Humans; Induced Pluripotent Stem Cells; Heart; Heterozygote; Homozygote; Phenotype; CRISPR-Cas Systems; COUP Transcription Factor II
PubMed: 38458031
DOI: 10.1016/j.scr.2024.103374 -
Biology Open Mar 2024Loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the...
Loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT), heterozygous (CDX2-Het), and homozygous null CDX2 (CDX2-KO) genotypes, differentiating these cells in a 2D gastruloid model, and subjecting these cells to single-nucleus RNA and ATAC sequencing, we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile, suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo, we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together, these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo.
Topics: Humans; CDX2 Transcription Factor; Cell Differentiation; Embryo, Mammalian; Gene Regulatory Networks; Induced Pluripotent Stem Cells; Mesoderm; Gene Dosage
PubMed: 38451093
DOI: 10.1242/bio.060323 -
Human Genomics Mar 2024Rare genetic disorders causing specific congenital developmental abnormalities often manifest in single families. Investigation of disease-causing molecular features are...
BACKGROUND/OBJECTIVES
Rare genetic disorders causing specific congenital developmental abnormalities often manifest in single families. Investigation of disease-causing molecular features are most times lacking, although these investigations may open novel therapeutic options for patients. In this study, we aimed to identify the genetic cause in an Iranian patient with severe skeletal dysplasia and to model its molecular function in zebrafish embryos.
RESULTS
The proband displays short stature and multiple skeletal abnormalities, including mesomelic dysplasia of the arms with complete humero-radio-ulna synostosis, arched clavicles, pelvic dysplasia, short and thin fibulae, proportionally short vertebrae, hyperlordosis and mild kyphosis. Exome sequencing of the patient revealed a novel homozygous c.374G > T, p.(Arg125Leu) missense variant in MSGN1 (NM_001105569). MSGN1, a basic-Helix-Loop-Helix transcription factor, plays a crucial role in formation of presomitic mesoderm progenitor cells/mesodermal stem cells during early developmental processes in vertebrates. Initial in vitro experiments show protein stability and correct intracellular localization of the novel variant in the nucleus and imply retained transcription factor function. To test the pathogenicity of the detected variant, we overexpressed wild-type and mutant msgn1 mRNA in zebrafish embryos and analyzed tbxta (T/brachyury/ntl). Overexpression of wild-type or mutant msgn1 mRNA significantly reduces tbxta expression in the tailbud compared to control embryos. Mutant msgn1 mRNA injected embryos depict a more severe effect, implying a gain-of-function mechanism. In vivo analysis on embryonic development was performed by clonal msgn1 overexpression in zebrafish embryos further demonstrated altered cell compartments in the presomitic mesoderm, notochord and pectoral fin buds. Detection of ectopic tbx6 and bmp2 expression in these embryos hint to affected downstream signals due to Msgn1 gain-of-function.
CONCLUSION
In contrast to loss-of-function effects described in animal knockdown models, gain-of-function of MSGN1 explains the only mildly affected axial skeleton of the proband and rather normal vertebrae. In this context we observed notochord bending and potentially disruption of pectoral fin buds/upper extremity after overexpression of msgn1 in zebrafish embryos. The latter might result from Msgn1 function on mesenchymal stem cells or on chondrogenesis in these regions. In addition, we detected ectopic tbx6 and bmp2a expression after gain of Msgn1 function in zebrafish, which are interconnected to short stature, congenital scoliosis, limb shortening and prominent skeletal malformations in patients. Our findings highlight a rare, so far undescribed skeletal dysplasia syndrome associated with a gain-of-function mutation in MSGN1 and hint to its molecular downstream effectors.
Topics: Animals; Female; Humans; Pregnancy; Abnormalities, Multiple; Dwarfism; Gain of Function Mutation; Iran; Osteochondrodysplasias; RNA, Messenger; T-Box Domain Proteins; Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 38448978
DOI: 10.1186/s40246-024-00593-w -
ELife Mar 2024The mammary gland is a unique organ that undergoes dynamic alterations throughout a female's reproductive life, making it an ideal model for developmental, stem cell and...
The mammary gland is a unique organ that undergoes dynamic alterations throughout a female's reproductive life, making it an ideal model for developmental, stem cell and cancer biology research. Mammary gland development begins in utero and proceeds via a quiescent bud stage before the initial outgrowth and subsequent branching morphogenesis. How mammary epithelial cells transit from quiescence to an actively proliferating and branching tissue during embryogenesis and, importantly, how the branch pattern is determined remain largely unknown. Here, we provide evidence indicating that epithelial cell proliferation and onset of branching are independent processes, yet partially coordinated by the Eda signaling pathway. Through heterotypic and heterochronic epithelial-mesenchymal recombination experiments between mouse mammary and salivary gland tissues and ex vivo live imaging, we demonstrate that unlike previously concluded, the mode of branching is an intrinsic property of the mammary epithelium whereas the pace of growth and the density of ductal tree are determined by the mesenchyme. Transcriptomic profiling and ex vivo and in vivo functional studies in mice disclose that mesenchymal Wnt/ß-catenin signaling, and in particular IGF-1 downstream of it critically regulate mammary gland growth. These results underscore the general need to carefully deconstruct the different developmental processes producing branched organs.
Topics: Mice; Animals; Epithelium; Epithelial Cells; Cell Proliferation; Morphogenesis; Wnt Signaling Pathway; Mesoderm; Mammary Glands, Animal
PubMed: 38441552
DOI: 10.7554/eLife.93326 -
Stem Cell Research & Therapy Mar 2024Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function,...
BACKGROUND
Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs).
METHODS
We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively.
RESULTS
iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators.
CONCLUSIONS
iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.
Topics: Humans; Pericytes; Induced Pluripotent Stem Cells; Becaplermin; Endothelin-1; Adenosine; Cell Proliferation
PubMed: 38433209
DOI: 10.1186/s13287-024-03671-x