Differentiation Of Progenitors Research Articles

Fibro-Adipogenic Progenitor Isolation, Expansion, and Differentiation from the Spiny Mouse Model

Fibro-Adipogenic Progenitor Isolation, Expansion, and Differentiation from the Spiny Mouse Model

Novel features of Drosophila hematopoiesis uncovered by long-term live imaging

Stem cells are subject to continuous regulation to ensure that the correct balance between stem cell differentiation and self-renewal is maintained. The dynamic and ongoing nature of stem cell regulation, as well as the complex signaling microenvironment in which stem cells are typically found, means that studying them in their endogenous environment in real time has multiple advantages over static fixed-sample approaches. We recently described a method for long-term, ex-vivo, live imaging of the blood progenitors in the Drosophila larval hematopoietic organ, the Lymph Gland (LG). This methodology has allowed us to analyze multiple aspects of fly hematopoiesis, in real time, in a manner that could not be carried out previously. Here, we describe novel insights derived from our quantitative live imaging approach. These insights include: the identification of extensive filopodia in the progenitors and description of their morphology and dynamics; visualization and quantitative analysis of JAK/STAT signaling in progenitors by the simultaneous tracking of thousands of vesicles containing internalized Domeless receptors; quantitative analysis of the location, morphology, and dynamics of mitochondria in blood progenitors; long-term tracking of patterns of cell division and migration of mature blood cell in the LG; long-term tracking of multiple cell behaviors in the distal committed progenitors; analysis of Ca2+ signaling of blood progenitors in the secondary lobes of the LG. Together, these observations illustrate the power of imaging fly hematopoiesis in real time and identify many previously undescribed processes and behaviors in the LG that are likely to play important roles in the regulation of progenitor differentiation and self-renewal.

CXCR4 signaling determines the fate of hematopoietic multipotent progenitors by stimulating mTOR activity and mitochondrial metabolism

Both cell-intrinsic and niche-derived, cell-extrinsic cues drive the specification of hematopoietic multipotent progenitors (MPPs) in the bone marrow, which comprise multipotent MPP1 cells and lineage-restricted MPP2, MPP3, and MPP4 subsets. Patients with WHIM syndrome, a rare congenital immunodeficiency caused by mutations that prevent desensitization of the chemokine receptor CXCR4, have an excess of myeloid cells in the bone marrow. Here, we investigated the effects of increased CXCR4 signaling on the localization and fate of MPPs. Knock-in mice bearing a WHIM syndrome–associated CXCR4 mutation ( CXCR4 1013 ) phenocopied the myeloid skewing of bone marrow in patients. Whereas MPP4 cells in wild-type mice differentiated into lymphoid cells, MPP4s in CXCR4 1013 knock-in mice differentiated into myeloid cells. This myeloid rewiring of MPP4s in CXCR4 1013 knock-in mice was associated with enhanced signaling mediated by the kinase mTOR and increased oxidative phosphorylation (OXPHOS). MPP4s also localized further from arterioles in the bone marrow of knock-in mice compared with wild-type mice, suggesting that the loss of extrinsic cues from the perivascular niche may also contribute to their myeloid skewing. Chronic treatment with the CXCR4 antagonist AMD3100 or the mTOR inhibitor rapamycin restored the lymphoid potential of MPP4s in knock-in mice. Thus, CXCR4 desensitization drives the lymphoid potential of MPP4 cells by dampening the mTOR-dependent metabolic changes that promote myeloid differentiation.

Dual role for Headcase in hemocyte progenitor fate determination in Drosophila melanogaster.

The hematopoietic organ of the Drosophila larva, the lymph gland, is a simplified representation of mammalian hematopoietic compartments, with the presence of hemocyte progenitors in the medullary zone (MZ), differentiated hemocytes in the cortical zone (CZ), and a hematopoietic niche called the posterior signaling centre (PSC) that orchestrates progenitor differentiation. Our previous work has demonstrated that the imaginal cell factor Headcase (Hdc, Heca) is required in the hematopoietic niche to control the differentiation of hemocyte progenitors. However, the downstream mechanisms of Hdc-mediated hematopoietic control remained unknown. Here we show that Hdc exerts this function by negatively regulating the insulin/mTOR signaling in the niche. When Hdc is depleted in the PSC, the overactivation of this pathway triggers reactive oxygen species (ROS) accumulation and, in turn, the differentiation of effector lamellocytes non-cell-autonomously. Although overactivation of insulin/mTOR signaling normally leads to an increase in the size of the hematopoietic niche, this effect is concealed by cell death caused by hdc loss-of-function. Moreover, we describe here that hdc silencing in progenitors causes cell-autonomous ROS elevation and JNK pathway activation, resulting in decreased MZ size and differentiation of lamellocytes. Similarly to the PSC niche, knocking down hdc in the MZ also leads to caspase activation. Notably, depleting Hdc in the progenitors triggers proliferation, an opposing effect to what is observed in the niche. These findings further our understanding of how progenitor maintenance in the larval lymph gland is controlled autonomously and non-cell-autonomously, and point towards new mechanisms potentially regulating HSC maintenance across vertebrates.

Zebrafish arterial valve development occurs through direct differentiation of second heart field progenitors.

Bicuspid Aortic Valve (BAV) is the most common congenital heart defect, affecting at least 2% of the population. The embryonic origins of BAV remain poorly understood, with few assays for validating patient variants, limiting the identification of causative genes for BAV. In both human and mouse, the left and right leaflets of the arterial valves arise from the outflow tract cushions, with interstitial cells originating from neural crest cells and the overlying endocardium through endothelial-to-mesenchymal transition (EndoMT). In contrast, an EndoMT-independent mechanism of direct differentiation of cardiac progenitors from the second heart field (SHF) is responsible for the formation of the anterior and posterior leaflets. Defects in either of these developmental mechanisms can result in BAV. Although zebrafish have been suggested as a model for human variant testing, their naturally bicuspid arterial valve has not been considered suitable for understanding human arterial valve development. Here, we have set out to investigate to what extent the processes involved in arterial valve development are conserved in zebrafish and ultimately, whether functional testing of BAV variants could be carried out. Using a combination of live imaging, immunohistochemistry and Cre-mediated lineage tracing, we show that the zebrafish arterial valve primordia develop directly from SHF progenitors with no contribution from EndoMT or neural crest, in keeping with the human and mouse anterior and posterior leaflets. Moreover, once formed, these primordia share common subsequent developmental events with all three aortic valve leaflets. Our work highlights a conserved ancestral mechanism of arterial valve leaflet formation from the SHF and identifies that development of the arterial valve is distinct from that of the atrioventricular valve in zebrafish. Crucially, this confirms the utility of zebrafish for understanding the development of specific BAV subtypes and arterial valve dysplasia, offering potential for high-throughput variant testing.

Regulating the formation of Müller glia-derived progenitor cells in the retina.

We summarize recent findings in different animal models regarding the different cell-signaling pathways and gene networks that influence the reprogramming of Müller glia into proliferating, neurogenic progenitor cells in the retina. Not surprisingly, most of the cell-signaling pathways that guide the proliferation and differentiation of embryonic retinal progenitors also influence the ability of Müller glia to become proliferating Müller glia-derived progenitor cells (MGPCs). Further, the neuronal differentiation of MGPC progeny is potently inhibited by networks of neurogenesis-suppressing genes in chick and mouse models but occurs freely in zebrafish. There are important differences between the model systems, particularly pro-inflammatory signals that are active in mature Müller glia in damaged rodent and chick retinas, but less so in fish retinas. These pro-inflammatory signals are required to initiate the process of reprogramming, but if sustained suppress the potential of Müller glia to become neurogenic MGPCs. Further, there are important differences in how activated Müller glia up- or downregulate pro-glial transcription factors in the different model systems. We review recent findings regarding regulatory cell signaling and gene networks that influence the activation of Müller glia and the transition of these glia into proliferating progenitor cells with neurogenic potential in fish, chick, and mouse model systems.

Interleukin-1β modulates lymphoid differentiation of Flt3-positive multipotent progenitors after transplantation

Myeloablative pre-conditioning facilitates the differentiation of transplanted hematopoietic stem and progenitor cells (HSPCs). However, the factors in the stress environment that regulate HSPC behavior remain elusive. Here, we investigated the mechanisms that shaped the cell fates of transplanted murine multipotent progenitors (MPPs) expressing the Fms-related receptor tyrosine kinase 3 gene (Flt3). Using lineage tracing, clonal analysis, and single-cell RNA sequencing (RNA-seq), we showed that the myeloablative environment increased lymphoid priming of Flt3+ MPPs and that their efficient B cell output required intact interleukin 1 (IL-1) signaling. The Flt3+ MPPs with short-term exposure to IL-1β underwent a myeloid-biased to lymphoid-biased cell fate switch and produced more lymphoid-biased progeny with a strongerB lymphopoiesis capacity invitro. Correspondingly, a brief exposure to IL-1β facilitated the B cell output of transplanted Flt3+ MPPs invivo. Together, our study demonstrated an unrecognized function of IL-1β in promoting B lymphopoiesis and highlighted a latent effect of IL-1β in regulating MPP cell fate dynamics.

Map3k1 suppresses terminal differentiation of migratory eye progenitors in planarian regeneration.

Proper stem cell targeting and differentiation is necessary for regeneration to succeed. In organisms capable of whole body regeneration, considerable progress has been made identifying wound signals initiating this process, but the mechanisms that control the differentiation of progenitors into mature organs are not fully understood. Using the planarian as a model system, we identify a novel function for map3k1, a MAP3K family member possessing both kinase and ubiquitin ligase domains, to negatively regulate terminal differentiation of stem cells during eye regeneration. Inhibition of map3k1 caused the formation of multiple ectopic eyes within the head, but without controlling overall head, brain, or body patterning. By contrast, other known regulators of planarian eye patterning like WntA and notum also regulate head regionalization, suggesting map3k1 acts distinctly. Eye resection and regeneration experiments suggest that unlike Wnt signaling perturbation, map3k1 inhibition did not shift the target destination of eye formation in the animal. Instead, map3k1(RNAi) ectopic eyes emerge in the regions normally occupied by migratory eye progenitors, and the onset of ectopic eyes after map3k1 inhibition coincides with a reduction to eye progenitor numbers. Furthermore, RNAi dosing experiments indicate that progenitors closer to their normal target are relatively more sensitive to the effects of map3k1, implicating this factors in controlling the site of terminal differentiation. Eye phenotypes were also observed after inhibition of map2k4, map2k7, jnk, and p38, identifying a putative pathway through which map3k1 prevents differentiation. Together, these results suggest that map3k1 regulates a novel control point in the eye regeneration pathway which suppresses the terminal differentiation of progenitors during their migration to target destinations.

Fetal Liver-like Organoids Recapitulate Blood-Liver Niche Development and Multipotent Hematopoiesis from Human Pluripotent Stem Cells.

The fetal liver is a hematopoietic organ, hosting a diverse and evolving progenitor population. While human liver organoids derived from pluripotent stem cells (PSCs) mimic aspects of embryonic and fetal development, they typically lack the complex hematopoietic niche and the interaction between hepatic and hematopoietic development. We describe the generation of human Fetal Liver-like Organoids (FLOs), that model human hepato-hematopoietic interactions previously characterized in mouse models. Developing FLOs first integrate a yolk sac-like hemogenic endothelium into hepatic endoderm and mesoderm specification. As the hepatic and hematopoietic lineages differentiate, the FLO culture model establishes an autonomous niche capable of driving subsequent progenitor differentiation without exogenous factors. Consistent with yolk sac-derived waves, hematopoietic progenitor cells (HPCs) within FLOs exhibit multipotency with a preference for myeloid lineage commitment, while retaining fetal B and T cell differentiation potential. We reconstruct in FLOs the embryonic monocyte-to-macrophage and granulocyte immune trajectories within the FLO microenvironment and assess their functional responses in the liver niche. In vivo, FLOs demonstrate a liver engraftment bias of hematopoietic cells, recapitulating a key phenomenon of human hematopoietic ontogeny. Our findings highlight the intrinsic capacity of liver organoids to support hematopoietic development, establishing FLOs as a platform for modeling and manipulating human blood-liver niche interactions during critical stages of development and disease.

Sex differences in the influence of adult-onset hypothyroidism on hippocampal progenitor survival and neuronal differentiation in mice.

The ongoing production of newborn neurons in the adult hippocampus is reported to be sensitive to perturbations of thyroid hormone signaling, in male rats and mice. Here, we examined whether the neurogenic changes evoked by adult-onset hypothyroidism exhibit sex differences, using male and female C57BL/6N mice. We assessed the impact of goitrogen-induced, adult-onset hypothyroidism on the postmitotic survival and differentiation of hippocampal progenitors in male and female mice. Adult-onset hypothyroidism evoked a significant decline in the postmitotic survival and neuronal differentiation of adult-born progenitors within the dentate gyrus hippocampal subfield of male, but not female, mice. We observed a significant decrease in the number of immature neurons within the hippocampi of adult-onset hypothyroid male mice, whereas adult-onset hypothyroidism evoked by goitrogens using the same treatment paradigms did not evoke any change in immature neuron number in female mice. Gene expression analysis within the hippocampi of euthyroid male and female mice revealed sex-dependent, differential expression of thyroid hormone receptor genes, as well as genes linked to thyroid hormone metabolism and transport. Collectively, our findings highlight sex differences in the influence of goitrogen-induced, adult-onset hypothyroidism on hippocampal neurogenesis, with male, but not female, mice exhibiting a decline in postmitotic hippocampal progenitor survival and neuronal differentiation. These findings underscore the importance of sex as a vital variable when considering the impact of thyroid hormone signaling on the adult hippocampal neurogenic niche.

PI3K/AKT signaling controls ICM maturation and proper epiblast and primitive endoderm specification in mice.

The inner cell mass (ICM) of early mouse embryos is specified into epiblast (Epi) and primitive endoderm (PrE) lineages during blastocyst formation. The antagonistic transcription factors (TFs) NANOG and GATA-binding protein 6 (GATA6) in combination with fibroblast growth factor (FGF)/extracellular-signal-regulated kinase (ERK) signaling are central actors in ICM fate choice. However, what initiates the specification of ICM progenitors into Epi or PrE and whether other factors are involved in this process has not been fully understood yet. Here, we show that phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) is constitutively active during preimplantation development. Using pharmacological inhibition, we demonstrate that PI3K/AKT enables the formation of a functional ICM capable of giving rise to both the Epi and the PrE: it maintains the expression of the TF NANOG, which specifies the Epi, and confers responsiveness to FGF4, which is essential for PrE specification. Our work thus identifies PI3K/AKT signaling as an upstream regulator controlling the molecular events required for both Epi and PrE specification.

Alternative Oxidase Expression Restores Kidney Progenitor Differentiation in Mice with Mitochondrial Complex lll Disruption

Alternative Oxidase Expression Restores Kidney Progenitor Differentiation in Mice with Mitochondrial Complex lll Disruption

Regulators of Intermediate Mesoderm Development Impact Neighboring Vessel Progenitor Specification

Regulators of Intermediate Mesoderm Development Impact Neighboring Vessel Progenitor Specification

Neuronal lineage tracing from progenitors in human cortical organoids reveals mechanisms of neuronal production, diversity, and disease

The contribution of progenitor subtypes to generating the billions of neurons produced during human cortical neurogenesis is not well understood. We developed the cortical organoid lineage-tracing (COR-LT) system for human cortical organoids. Differential fluorescent reporter activation in distinct progenitor cells leads to permanent reporter expression, enabling the progenitor cell lineage of neurons to be determined. Surprisingly, nearly all excitatory neurons produced in cortical organoids were generated indirectly from intermediate progenitor cells. Additionally, neurons of different progenitor lineages were transcriptionally distinct. Isogenic lines made from an autistic individual with and without a likely pathogenic CTNNB1 variant demonstrated that the variant substantially altered the proportion of neurons derived from specific progenitor cell lineages, as well as the lineage-specific transcriptional profiles of these neurons, suggesting a pathogenic mechanism for this mutation. These results suggest individual progenitor subtypes play roles in generating the diverse neurons of the human cerebral cortex.

BMP Antagonist Gremlin 2 Regulates Hippocampal Neurogenesis and Is Associated with Seizure Susceptibility and Anxiety.

The Bone Morphogenetic Protein (BMP) signaling pathway is vital in neural progenitor cell proliferation, specification, and differentiation. The BMP signaling antagonist Gremlin 2 (Grem2) is the most potent natural inhibitor of BMP expressed in the adult brain; however its function remains unknown. To address this knowledge gap, we have analyzed mice lacking Grem2 via homologous recombination (Grem2-/- ). Histological analysis of brain sections revealed significant scattering of CA3 pyramidal cells within the dentate hilus in the hippocampus of Grem2-/- mice. Furthermore, the number of proliferating neural stem cells and neuroblasts was significantly decreased in the subgranular zone of Grem2-/- mice compared with that of wild-type (WT) controls. Due to the role of hippocampal neurogenesis in neurological disorders, we tested mice on a battery of neurobehavioral tests. Grem2-/- mice exhibited increased anxiety on the elevated zero maze in response to acute and chronic stress. Specifically, male Grem2-/- mice showed increased anxiogenesis following chronic stress, and this was correlated with higher levels of BMP signaling and decreased proliferation in the dentate gyrus. Additionally, when chemically challenged with kainic acid, Grem2-/- mice displayed a higher susceptibility to and increased severity of seizures compared with WTs. Together, our data indicate that Grem2 regulates BMP signaling and is vital in maintaining homeostasis in adult hippocampal neurogenesis and structure. Furthermore, the lack of Grem2 contributes to the development and progression of neurogenesis-related disorders such as anxiety and epilepsy.

Genomic mosaicism reveals developmental organization of trunk neural crest-derived ganglia.

The neural crest generates numerous cell types, but conflicting results leave developmental origins unresolved. Here using somatic mosaic variants as cellular barcodes, we infer embryonic clonal dynamics of trunk neural crest, focusing on the sensory and sympathetic ganglia. From three independent adult neurotypical human donors, we identified 1,278 mosaic variants using deep whole-genome sequencing, then profiled allelic fractions in 187 anatomically dissected ganglia. We found a massive rostrocaudal spread of progenitor clones specific to sensory or sympathetic ganglia, which unlike in the brain, showed robust bilateral distributions. Computational modeling suggested neural crest progenitor fate specification preceded delamination from neural tube. Single-cell multiomic analysis suggested both neurons and glia contributed to the rostrocaudal clonal organization. CRISPR barcoding in mice and live imaging in quail embryos confirmed these clonal dynamics across multiple somite levels. Our findings reveal an evolutionarily conserved clonal spread of cells populating peripheral neural ganglia.

Generation of ex vivo autologous hematopoietic stem cell-derived T lymphocytes for cancer immunotherapy

CD19CAR-T cell therapy demonstrated promising outcomes in relapsed/refractory B-cell malignancies. Nonetheless, the limited T-cell function and ineffective T-cell apheresis for therapeutic purposes are still concern in heavily pretreated patients. We investigated the feasibility of generating hematopoietic stem cell-derived T lymphocytes (HSC-T) for cancer immunotherapy. The patients’ autologous peripheral blood HSCs were enriched for CD34+ and CD3+ cells. The CD34+ cells were then cultured following three steps of lymphoid progenitor differentiation, T-cell differentiation, and T-cell maturation processes. HSC-T cells were successfully generated with robust fold expansion of 3735 times. After lymphoid progenitor differentiation, CD5+ and CD7+ cells remarkably increased (65–84 %) while CD34+ cells consequentially declined. The mature CD3+ cells were detected up to 40 % and 90 % on days 42 and 52, respectively. The majority of HSC-T population was naïve phenotype compared to CD3-T cells (73 % vs 34 %) and CD8:CD4 ratio was 2:1. The higher level of cytokine and cytotoxic granule secretion in HSC-T was observed after activation. HSC-T cells were assessed for clinical application and found that CD19CAR-transduced HSC-T cells demonstrated higher cytokine secretion and a trend of superior cytotoxicity against CD19+ target cells compared to control CAR-T cells. A chronic antigen stimulation assay revealed similar T-cell proliferation, stemness, and exhaustion phenotypes among CAR-T cell types. In conclusions, autologous HSC-T was feasible to generate with preserved T-cell efficacy. The HSC-T cells are potentially utilized as an alternative option for cellular immunotherapy.

P300/CBP KATs Are Critical for Maturation and Differentiation of Adult Neural Progenitors.

Epigenetic modifications play a pivotal role in the process of neurogenesis. Among these modifications, reversible acetylation fine-tunes gene expression for both embryonic and adult neurogenesis. The CBP/KAT3A and its paralogue p300/KAT3B are well-known lysine acetyltransferases with transcriptional coactivation ability that engage in neural plasticity and memory. The exclusive role of their KAT activity in neurogenesis and memory could not be addressed due to the absence of a p300/CBP modulator, which can cross the blood-brain barrier. Previous work from our laboratory has shown that a small molecule activator, TTK21, specific to CBP/p300, when conjugated to glucose-derived carbon nanospheres (CSP), is efficiently delivered to the mouse brain and could induce dendritic branching and extend long-term memory. However, the molecular mechanisms of p300 acetyltransferase activity-dependent enhanced dendritogenesis are yet to be understood. Here, we report that CSP-TTK21 treatment to primary neuronal culture derived from mouse embryo enhances the expression of five critical genes: Neurod1 (central nervous system development), Tubb3 (immature neural marker), Camk2a (synaptic plasticity and LTP), Snap25 (spine morphogenesis plasticity), and Scn2a (propagation of the action potential). Activation of these genes by inducing the p300/CBP KAT activity presumably promotes the maturation and differentiation of adult neuronal progenitors and thereby the formation of long and highly branched doublecortin-positive functional neurons in the subgranular zone of the dentate gyrus.

Pax6 expressing neuroectodermal and ocular stem cells: Its role from a developmental biology perspective.

Pax-6 emerges as a critical transcription factor that guides the fate of stem cells towards neural lineages. Its expression influences the differentiation of neural progenitors into diverse neuronal subtypes, glial cells, and other neural cell types. Pax-6 operates with other regulatory factors to ensure the precise patterning and organization of the developing nervous system. The intricate interplay between Pax-6 and other signaling pathways, transcription factors, and epigenetic modifiers underpins the complicated balance between stem cell maintenance, proliferation, and differentiation in neuroectodermal and ocular contexts. Dysfunction of Pax-6 can lead to a spectrum of developmental anomalies, underscoring its importance in these processes. This review highlights the essential role of Pax-6 expression in neuroectodermal and ocular stem cells, shedding light on its significance in orchestrating the intricate journey from stem cell fate determination to the emergence of diverse neural and ocular cell types. The comprehensive understanding of Pax-6 function gained from a developmental biology perspective offers valuable insights into normal development and potential therapeutic avenues for neuroectodermal and ocular disorders.

The Effect of Resveratrol on Gamma Globin Gene Expression in Patients with Beta Thalassemia: The Role of Adaptation to Cellular Stress

HbF induction is an appropriate strategy to ameliorate the severity of β-thalassemia symptoms. Hydroxyurea (HU) is the most common chemical agent introduced as an HbF inducer but responsiveness to HU is variable and the introduction of HbF inducers alternative to HU with low cytotoxicity has been a crucial challenge. Resveratrol is an HbF inducer agent that may have favorable effects on the differentiation of hematopoietic erythroid progenitors (HEPs). The present study aimed to investigate the effect of resveratrol on γ-globin, stress response, and anti-apoptotic gene expression among hydroxyurea (HU)-responders and HU-nonresponders (HU-NR). Four cases of HU-R and four cases of HU-NR were studied. HEPs of the patients were cultured, and the expression of γ-globin, Foxo3, and Bclxl was assessed. Moreover, the differentiation and apoptotic rate of the cells were investigated using flow cytometry analysis. In three cases, the γ-globin gene expression increased after resveratrol treatment. All of the HU-NR patients were also non-responders to resveratrol (Res-NR). The expression of Foxo3 and Bclxl genes was higher in responders to resveratrol (Res-R) compared to non-responders (Res-NR). The rate of apoptosis in Res-R patients was also lower than in Res-NR. Responders to resveratrol also had a higher rate of HEP maturation. The cells of both HU–NR and Res-NR patients could not adapt to stress conditions and proceed to the erythroid differentiation. In conclusion, resveratrol increased the γ-globin expression in HEPs of β-thalassemia patients. The response was observed only in R-HU patients with similar cellular pathways.