Progenitor Cell Line Research Articles

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.

High-Throughput Assessment of Metabolism-Mediated Neurotoxicity by Co-Culture of Neurospheres and Liver Spheroids.

The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high-throughput, metabolism-mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three-dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384-well plate and for cultivating ReNcell VM in 3D on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384-well plate containing HepaRG spheroids and then tested with 3D-cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC50 values for each compound, thus evaluating metabolism-mediated neurotoxicity. © 2024 Wiley Periodicals LLC. Basic Protocol 1: HepaRG spheroid culture in an ultralow attachment (ULA) 384-well plate and the assessment of drug-metabolizing enzyme (DME) activities Basic Protocol 2: 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism-mediated neurotoxicity Basic Protocol 3: Image acquisition, processing, and data analysis.

Selective Induction of Renal Interstitial Progenitor Cell Lineages from Human-Induced Pluripotent Stem Cells (hiPSCs) for Understanding of Mesangial and Erythropoietin (EPO)-Producing Cell Development and Kidney Regeneration

Selective Induction of Renal Interstitial Progenitor Cell Lineages from Human-Induced Pluripotent Stem Cells (hiPSCs) for Understanding of Mesangial and Erythropoietin (EPO)-Producing Cell Development and Kidney Regeneration

Ketamine Prevents Inflammation-Induced Reduction of Human Hippocampal Neurogenesis via Inhibiting the Production of Neurotoxic Metabolites of the Kynurenine Pathway.

Understanding the precise mechanisms of ketamine is crucial for replicating its rapid antidepressant effects without inducing psychomimetic changes. Here, we explore whether the antidepressant-like effects of ketamine enantiomers are underscored by protection against cytokine-induced reductions in hippocampal neurogenesis and activation of the neurotoxic kynurenine pathway in our well-established in vitro model of depression in a dish. We used the fetal hippocampal progenitor cell line (HPC0A07/03C) to investigate ketamine's impact on cytokine-induced reductions in neurogenesis in vitro. Cells were treated with interleukin- 1beta (IL-1b) (10ng/mL) or IL-6 (50 pg/mL), alone or in combination with ketamine enantiomers arketamine (R-ketamine, 400nM) or esketamine (S-ketamine, 400nM) or antidepressants sertraline (1mM) or venlafaxine (1mM). Resembling the effect of antidepressants, both ketamine enantiomers prevented IL-1b- and IL-6-induced reduction in neurogenesis and increase in apoptosis. This was mediated by inhibition of IL-1b-induced production of IL-2 and IL-13 by R-ketamine and of IL-1b-induced tumor necrosis factor-alpha by S-ketamine. Likewise, R-ketamine inhibited IL-6-induced production of IL-13, whereas S-ketamine inhibited IL-6-induced IL-1b and IL-8. Moreover, both R- and S-ketamine prevented IL-1b-induced increases in indoleamine 2,3-dioxygenase expression as well as kynurenine production, which in turn was shown to mediate the detrimental effects of IL-1b on neurogenesis and apoptosis. In contrast, neither R- nor S-ketamine prevented IL-6-induced kynurenine pathway activation. Results suggest that R- and S-ketamine have pro-neurogenic and anti-inflammatory properties; however, this is mediated by inhibition of the kynurenine pathway only in the context of IL-1b. Overall, this study enhances our understanding of the mechanisms underlying ketamine's antidepressant effects in the context of different inflammatory phenotypes, ultimately leading to the development of more effective, personalized therapeutic approaches for patients suffering from depression.

CpG Methylation of Receptor Activator NF-κB (RANK) Gene Promoter Region Delineates Senescence-Related Decrease of RANK Gene Expression.

While the rapid decrease in estrogen is well known as the main cause of postmenopausal osteoporosis in women, the precise pathogenesis of senile osteoporosis in the elderly regardless of gender is largely unknown. The age-related epigenetic regulation of receptor activator NF-κB (RANK) gene expression was investigated with the use of a high-passaged mouse osteoclast progenitor cell line, RAW264.7, as an in vitro model of aging. In the RAW264.7 cells after repeated passages, receptor RANK expression was downregulated, resulting in decreased soluble RANK ligand (sRANKL)-induced osteoclastogenesis, expression of tartrate-resistant acid phosphatase-5b (TRAcP) and cathepsin K (CTSK). Methylation-specific PCR and bisulfite mapping revealed hypermethylation of CpG-loci located in the RANK gene promoter in multiple-passaged cells. ICON probe-mediated in situ assessment of methylated-cytosine at the CpG loci revealed an increase in the percentage of methylated RAW264.7 cells in the RANK gene in a passage-dependent manner. Conversely, upon treatment with demethylating agent 5-aza-2-deoxycytidine (5-aza-dC), high-passaged RAW264.7 cells displayed restored expression of the RANK gene, osteoclastogenesis, TRAcP and CTSK. Ex vivo cultures of splenic macrophages from young (10.5 W) and aged (12 M) mice also showed that CpG methylation was predominant in the aged animals, resulting in reduced RANK expression and osteoclastogenesis. Reduced RANK expression by age-related accumulation of DNA methylation, albeit in a limited population of osteoclast precursor cells, might be, at least in part, indicative of low-turnover bone characteristic of senile osteoporosis.

A cellular reporter system to evaluate endogenous fetal hemoglobin induction and screen for therapeutic compounds.

Reactivation of fetal hemoglobin expression alleviates the symptoms associated with β-globinopathies, severe hereditary diseases with significant global health implications due to their high morbidity and mortality rates. The symptoms emerge following the postnatal transition from fetal-to-adult hemoglobin expression. Extensive research has focused on inducing the expression of the fetal γ-globin subunit to reverse this switch and ameliorate these symptoms. Despite decades of research, only one compound, hydroxyurea, found its way to the clinic as an inducer of fetal hemoglobin. Unfortunately, its efficacy varies among patients, highlighting the need for more effective treatments. Erythroid cell lines have been instrumental in the pursuit of both pharmacological and genetic ways to reverse the postnatal hemoglobin switch. Here, we describe the first endogenously tagged fetal hemoglobin reporter cell line based on the adult erythroid progenitor cell line HUDEP2. Utilizing CRISPR-Cas9-mediated knock-in, a bioluminescent tag was integrated at the HBG1 gene. Subsequent extensive characterization confirmed that the resulting reporter cell line closely mirrors the HUDEP2 characteristics and that the cells report fetal hemoglobin induction with high sensitivity and specificity. This novel reporter cell line is therefore highly suitable for evaluating genetic and pharmacologic strategies to induce fetal hemoglobin. Furthermore, it provides an assay compatible with high-throughput drug screening, exemplified by the identification of a cluster of known fetal hemoglobin inducers in a pilot study. This new tool is made available to the research community, with the aspiration that it will accelerate the search for safer and more effective strategies to reverse the hemoglobin switch.

Human Neutrophil Elastase: Characterization of Intra- vs. Extracellular Inhibition.

Neutrophil elastase (HNE), like other members of the so-called GASPIDs (Granule-Associated Serine Peptidases of Immune Defense), is activated during protein biosynthesis in myeloid precursors and stored enzymatically active in cytoplasmic granules of resting neutrophils until secreted at sites of host defense and inflammation. Inhibitors thus could bind to the fully formed active site of the protease intracellularly in immature progenitors, in circulating neutrophils, or to HNE secreted into the extracellular space. Here, we have compared the ability of a panel of diverse inhibitors to inhibit HNE in the U937 progenitor cell line, in human blood-derived neutrophils, and in solution. Most synthetic inhibitors and, surprisingly, even a small naturally occurring proteinaceous inhibitor inhibit HNE intracellularly, but the extent and dynamics differ markedly from classical enzyme kinetics describing extracellular inhibition. Intracellular inhibition of HNE potentially affects neutrophil functions and has side effects, but it avoids competition of inhibitors with extracellular substrates that limit its efficacy. As both intra- and extracellular inhibition have advantages and disadvantages, the quantification of intracellular inhibition, in addition to classical enzyme kinetics, will aid the design of novel, clinically applicable HNE inhibitors with targeted sites of action.

Generation of musculoskeletal cells from human urine epithelium-derived presomitic mesoderm cells

BackgroundNumerous studies have shown that somite development is a necessary stage of myogenesis chondrogenesis and osteogenesis. Our previous study has established a stable presomitic mesoderm progenitor cell line (UiPSM) in vitro. Naturally, we wanted to explore whether UiPSM cell can develop bone and myogenic differentiation.ResultsSelective culture conditions yielded PAX3 and PAX7 positive skeletal muscle precursors from UiPSM cells. The skeletal muscle precursors undergo in vitro maturation resulting in myotube formation. MYOD effectively promoted the maturity of the skeletal myocytes in a short time. We found that UiPSM and MYOD mediated UiPSM cell-derived skeletal myocytes were viable after transplantation into the tibialis anterior muscle of MITRG mice, as assessed by bioluminescence imaging and scRNA-seq. Lack of teratoma formation and evidence of long-term myocytes engraftment suggests considerable potential for future therapeutic applications. Moreover, UiPSM cells can differentiate into osteoblast and chondroblast cells in vitro.ConclusionsUiPSM differentiation has potential as a developmental model for musculoskeletal development research and treatment of musculoskeletal disorders.

Characterization of immortalized bone marrow erythroid progenitor adult (imBMEP-A)—The first inducible immortalized red blood cell progenitor cell line derived from bone marrow CD71-positive cells

Background aimsEx vivo production of red blood cells (RBCs) represents a promising alternative for transfusion medicine. Several strategies have been described to generate erythroid cell lines from different sources, including embryonic, induced pluripotent, and hematopoietic stem cells. All these approaches have in common that they require elaborate differentiation cultures whereas the yield of enucleated RBCs is inefficient. MethodsWe generated a human immortalized adult erythroid progenitor cell line derived from bone marrow CD71-positive erythroid progenitor cells (immortalized bone marrow erythroid progenitor adult, or imBMEP-A) by an inducible expression system, to shorten differentiation culture necessary for terminal erythroid differentiation. It is the first erythroid cell line that is generated from direct reticulocyte progenitors and demonstrates robust hemoglobin production in the immortalized state. ResultsMorphologic analysis of the immortalized cells showed that the preferred cell type of the imBMEP-A line corresponds to hemoglobin-producing basophilic erythroblasts. In addition, we were able to generate a stable cell line from a single cell clone with the triple knockout of RhAG, RhDCE and KELL. After removal of doxycycline, part of the cells differentiated into normoblasts and reticulocytes within 5–7 days. ConclusionsOur results demonstrate that the imBMEP-A cell line can serve as a stable and straightforward modifiable platform for RBC engineering in the future.

Expansion and differentiation of human neural stem cells on synthesized integrin binding peptide surfaces

The extracellular matrix plays a crucial role in the growth of human neural stem cells (hNSCs) by forming a stem cell niche, both in vitro and in vivo. The demand for defined synthetic substrates has been increasing recently in stem cell research, reflecting the requirements for precise functions and safety concerns in potential clinical approaches. In this study, we tested the adhesion and expansion of one of the most representative hNSC lines, the ReNcell VM Human Neural Progenitor Cell Line, in a pure-synthesized short peptide-based in vitro niche using a previously established integrin-binding peptide array. Spontaneous cell differentiation was then induced using two different in vitro approaches to further confirm the multipotent features of cells treated with the peptides. Twelve different integrin-binding peptides were capable of supporting hNSC adhesion and expansion at varied proliferation rates. In the ReNcell medium-based differentiation approach, cells detached in almost all peptide-based groups, except integrin α5β1 binding peptide. In an altered differentiation process induced by retinoic acid containing neural differentiation medium, cell adhesion was retained in all 12 peptide groups. These peptides also appeared to have varied effects on the differentiation potential of hNSCs towards neurons and astrocytes. Our findings provide abundant options for the development of in vitro neural stem cell niches and will help develop promising tools for disease modeling and future stem cell therapies for neurological diseases.

Defective flow space limits the scaling up of turbulence bioreactors for platelet generation

To complement donor-dependent platelets supplies, we previously developed an ex vivo manufacturing system using induced pluripotent stem cell (iPSC)-derived expandable immortalized megakaryocyte progenitor cell lines (imMKCLs), and a turbulent flow bioreactor to generate iPSC-derived platelets products (iPSC-PLTs). However, the tank size of the bioreactor was limited to 10 L. Here we examined the feasibility of scaling up to 50 L with reciprocal motion by two impellers. Under optimized turbulence parameters corresponding to 10 L bioreactor, 50 L bioreactor elicited iPSC-PLTs with intact in vivo hemostatic function but with less production efficiency. This insufficiency was caused by increased defective turbulent flow space. A computer simulation proposed that designing 50 L turbulent flow bioreactor with three impellers or a new bioreactor with a modified rotating impeller and unique structure reduces this space. These findings indicate that large-scale iPSC-PLTs manufacturing from cultured imMKCLs requires optimization of the tank structure in addition to optimal turbulent energy and shear stress.

SFlt-1 impairs neurite growth and neuronal differentiation in SH-SY5Y cells and human neurons.

Pre-eclampsia (PE) is a hypertensive disorder of pregnancy which is associated with increased risk of neurodevelopmental disorders in exposed offspring. The pathophysiological mechanisms mediating this relationship are currently unknown, and one potential candidate is the anti-angiogenic factor soluble Fms-like tyrosine kinase 1 (sFlt-1), which is highly elevated in PE. While sFlt-1 can impair angiogenesis via inhibition of VEGFA signalling, it is unclear whether it can directly affect neuronal development independently of its effects on the vasculature. To test this hypothesis, the current study differentiated the human neural progenitor cell (NPC) line ReNcell® VM into a mixed culture of mature neurons and glia, and exposed them to sFlt-1 during development. Outcomes measured were neurite growth, cytotoxicity, mRNA expression of nestin, MBP, GFAP, and βIII-tubulin, and neurosphere differentiation. sFlt-1 induced a significant reduction in neurite growth and this effect was timing- and dose-dependent up to 100 ng/ml, with no effect on cytotoxicity. sFlt-1 (100 ng/ml) also reduced βIII-tubulin mRNA and neuronal differentiation of neurospheres. Undifferentiated NPCs and mature neurons/glia expressed VEGFA and VEGFR-2, required for endogenous autocrine and paracrine VEGFA signalling, while sFlt-1 treatment prevented the neurogenic effects of exogenous VEGFA. Overall, these data provide the first experimental evidence for a direct effect of sFlt-1 on neurite growth and neuronal differentiation in human neurons through inhibition of VEGFA signalling, clarifying our understanding of the potential role of sFlt-1 as a mechanism by which PE can affect neuronal development.

A multi-omic single-cell landscape of cellular diversification in the developing human cerebral cortex

The vast neuronal diversity in the human neocortex is vital for high-order brain functions, necessitating elucidation of the regulatory mechanisms underlying such unparalleled diversity. However, recent studies have yet to comprehensively reveal the diversity of neurons and the molecular logic of neocortical origin in humans at single-cell resolution through profiling transcriptomic or epigenomic landscapes, owing to the application of unimodal data alone to depict exceedingly heterogeneous populations of neurons. In this study, we generated a comprehensive compendium of the developing human neocortex by simultaneously profiling gene expression and open chromatin from the same cell. We computationally reconstructed the differentiation trajectories of excitatory projection neurons of cortical origin and inferred the regulatory logic governing lineage bifurcation decisions for neuronal diversification. We demonstrated that neuronal diversity arises from progenitor cell lineage specificity and postmitotic differentiation at distinct stages. Our data paves the way for understanding the primarily coordinated regulatory logic for neuronal diversification in the neocortex.

Molecular Characterization and Inhibition of a Novel Stress-Induced Mitochondrial Protecting Role for Misfolded TrkAIII in Human SH-SY5Y Neuroblastoma Cells.

Pediatric neuroblastomas (NBs) are heterogeneous, aggressive, therapy-resistant embryonal tumors that originate from cells of neural crest origin committed to the sympathoadrenal progenitor cell lineage. Stress- and drug-resistance mechanisms drive post-therapeutic relapse and metastatic progression, the characterization and inhibition of which are major goals in improving therapeutic responses. Stress- and drug-resistance mechanisms in NBs include alternative TrkAIII splicing of the neurotrophin receptor tropomyosin-related kinase A (NTRK1/TrkA), which correlates with post-therapeutic relapse and advanced-stage metastatic disease. The TrkAIII receptor variant exerts oncogenic activity in NB models by mechanisms that include stress-induced mitochondrial importation and activation. In this study, we characterize novel targetable and non-targetable participants in this pro-survival mechanism in TrkAIII-expressing SH-SY5Y NB cells, using dithiothreitol (DTT) as an activator and a variety of inhibitors by regular and immunoprecipitation Western blotting of purified mitochondria and IncuCyte cytotoxicity assays. We report that stress-induced TrkAIII misfolding initiates this mechanism, resulting in Grp78, Ca2+-calmodulin, adenosine ribosylating factor (Arf) and Hsp90-regulated mitochondrial importation. TrkAIII imported into inner mitochondrial membranes is cleaved by Omi/high temperature requirement protein A2 (HtrA2) then activated by a mechanism dependent upon calmodulin kinase II (CaMKII), alpha serine/threonine kinase (Akt), mitochondrial Ca2+ uniporter and reactive oxygen species (ROS), involving inhibitory mitochondrial protein tyrosine phosphatase (PTPase) oxidation, resulting in phosphoinositide 3 kinase (PI3K) activation of mitochondrial Akt, which enhances stress resistance. This novel pro-survival function for misfolded TrkAIII mitigates the cytotoxicity of mitochondrial Ca2+ homeostasis disrupted during integrated stress responses, and is prevented by clinically approved Trk and Akt inhibitors and also by inhibitors of 78kDa glucose regulated protein (Grp78), heat shock protein 90 (Hsp90), Ca2+-calmodulin and PI3K. This identifies Grp78, Ca2+-calmodulin, Hsp90, PI3K and Akt as novel targetable participants in this mechanism, in addition to TrkAIII, the inhibition of which has the potential to enhance the stress-induced elimination of TrkAIII-expressing NB cells, with the potential to improve therapeutic outcomes in NBs that exhibit TrkAIII expression and activation.

20 Large Burn Injury Leads to Pathological Alteration of Hematopoietic Stem and Progenitor Cell Lineage Commitment

20 Large Burn Injury Leads to Pathological Alteration of Hematopoietic Stem and Progenitor Cell Lineage Commitment

Multiple parallel cell lineages in the developing mammalian cerebral cortex.

Cortical neurogenesis follows a simple lineage: apical radial glia cells (RGCs) generate basal progenitors, and these produce neurons. How this occurs in species with expanded germinal zones and a folded cortex, such as human, remains unclear. We used single-cell RNA sequencing from individual cortical germinal zones in ferret and barcoded lineage tracking to determine the molecular diversity of progenitor cells and their lineages. We identified multiple RGC classes that initiate parallel lineages, converging onto a common class of newborn neuron. Parallel RGC classes and transcriptomic trajectories were repeated across germinal zones and conserved in ferret and human, but not in mouse. Neurons followed parallel differentiation trajectories in the gyrus and sulcus, with different expressions of human cortical malformation genes. Progenitor cell lineage multiplicity is conserved in the folded mammalian cerebral cortex.

Lack of Elevated Expression of TGFβ3 Contributes to the Delay of Epithelial Wound Healing in Diabetic Corneas.

To investigate the mechanisms underlying the differential roles of TGFβ1 and TGFβ3 in accelerating corneal epithelial wound healing (CEWH) in diabetic (DM) corneas, with normoglycemia (NL) corneas as the control. Two types of diabetic mice, human corneal organ cultures, mouse corneal epithelial progenitor cell lines, and bone marrow-derived macrophages (BMDMs) were employed to assess the effects of TGFβ1 and TGFβ3 on CEWH, utilizing quantitative PCR, western blotting, ELISA, and whole-mount confocal microscopy. Epithelial debridement led to an increased expression of TGFβ1 and TGFβ3 in cultured human NL corneas, but only TGFβ1 in DM corneas. TGFβ1 and TGFβ3 inhibition was significantly impeded, but exogenous TGFβ1 and, more potently, TGFβ3 promoted CEWH in cultured TKE2 cells and in NL and DM C57BL6 mouse corneas. Wounding induced similar levels of p-SMAD2/SMAD3 in NL and DM corneas but weaker ERK1/2, Akt, and EGFR phosphorylation in DM corneas compared to NL corneas. Whereas TGFβ1 augmented SMAD2/SMAD3 phosphorylation, TGFβ3 preferentially activated ERK, PI3K, and EGFR in healing DM corneas. Furthermore, TGFβ1 and TGFβ3 differentially regulated the expression of S100a9, PAI-1, uPA/tPA, and CCL3 in healing NL and DM corneas. Finally, TGFβ1 induced the expression of M1 macrophage markers iNOS, CD86, and CTGF, whereas TGFβ3 promoted the expression of M2 markers CD206 and NGF in BMDMs from db/db or db/+ mice. Hyperglycemia disrupts the balanced expression of TGFβ3/TGFβ1, resulting in delayed CEWH, including impaired sensory nerve regeneration in the cornea. Supplementing TGFβ3 in DM wounds may hold therapeutic potential for accelerating delayed wound healing in diabetic patients.

Abstract 2920: In vitro assays for investigating the effects of cancer therapeutics on bone

Abstract Bone microenvironment is an important factor in regulating bone metastasis. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Stimulatory factors released from the resorbed bone matrix in turn promote proliferation of cancer cells and this process, referred as the vicious cycle, often leads to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. The vicious cycle observed in osteolytic disease occurs also in osteoblastic metastases, and in addition cancer cells produce osteoblast-stimulating factors. Cancers with bone metastases often include both osteolytic and osteoblastic lesions. Our aim was to establish in vitro cell culture models to study the effects of cancer therapeutics on osteoblast and osteoclast differentiation and activity. In the osteoclast differentiation assay, human osteoclast precursor cells (Lonza) were cultured on bovine bone slices for 7 days. Denosumab was added in the cultures at day 0, and tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) activity was measured from the culture medium collected at day 7. Osteoclast activity was studied by allowing osteoclasts resorb bone after 7 days’ maturation period for additional 3 days in the presence of odanacatib. The amount of C-terminal cross-linked telopeptides of type I collagen (CTX-I) was measured in the culture medium collected at day 10 to quantitate osteoclast activity. Denosumab and odanacatib showed strong concentration dependent inhibition of osteoclast differentiation and activity, respectively. A mouse osteoblast progenitor cell line KS483 (Pharmatest Services) was used to study differentiation and activity of osteoblasts with test compounds bone morphogenetic protein 2 (BMP-2) and estradiol (E2). In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8. In the osteoblast activity assay, the cells were cultured for 13 days. N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to indicate the effects on organic bone formation and calcium deposition was determined at the end of the study as a marker of inorganic bone formation. BMP-2 and E2 stimulated osteoblast differentiation and activity indicated by the increase in ALP activity, PINP and calcium levels. We conclude that these culture systems can be used for studying the effects of cancer therapeutics and identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. Citation Format: Jenni H. Mäki-Jouppila, Mervi Ristola, Jukka Rissanen, Katja Fagerlund. In vitro assays for investigating the effects of cancer therapeutics on bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2920.

Amphiregulin Switches Progenitor Cell Fate for Lineage Commitment During Gastric Mucosal Regeneration

Background & AimsIsthmic progenitors, tissue-specific stem cells in the stomach corpus, maintain mucosal homeostasis by balancing between proliferation and differentiation to gastric epithelial lineages. The progenitor cells rapidly adopt an active state in response to mucosal injury. However, it remains unclear how the isthmic progenitor cell niche is controlled during the regeneration of damaged epithelium. MethodsWe recapitulated tissue recovery process after acute mucosal injury in the mouse stomach. BrdU incorporation was utilized to trace newly generated cells during the injury and recovery phases. To define epithelial lineage commitment process during recovery, we performed single cell RNA-sequencing (scRNA-seq) on epithelial cells from the mouse stomachs. We validated the effects of amphiregulin (AREG) on mucosal recovery, utilizing recombinant AREG treatment or AREG deficient mice. ResultsWe determined that an EGFR ligand, AREG, can control progenitor cell lineage commitment. Based on the identification of lineage-committed subpopulations in the corpus epithelium through scRNA-seq and BrdU incorporation, we showed that isthmic progenitors mainly transition into short-lived surface cell lineages, but are less frequently committed to long-lived parietal cell lineages in homeostasis. However, mucosal regeneration after damage directs the lineage commitment of isthmic progenitors towards parietal cell lineages. During recovery, AREG treatment promoted repopulation with parietal cells, while suppressing surface cell commitment of progenitors. In contrast, TGF-alpha did not alter parietal cell regeneration, but did induce expansion of surface cell populations. AREG deficiency impairs parietal cell regeneration, but increases surface cell commitment. ConclusionsThese data demonstrate that different EGF receptor ligands can distinctly regulate isthmic progenitor-driven mucosal regeneration and lineage commitment.

Cementocyte-derived extracellular vesicles regulate osteoclastogenesis and osteoblastogenesis

Background/purposeCementum shares many properties with bone; however, in contrast to bone, it is not innervated or vascularized and has a limited capacity for remodeling. Osteocytes located in the lacunae-canalicular system of bone tissue play a central role in bone remodeling by communicating with osteoblasts and osteoclasts. Although cementocytes are present in cellular cementum and are morphologically similar to osteocytes, it remains unclear whether they are involved in the dynamic functional regulation of metabolism in cementum. The present study focused on the extracellular vesicles (EVs) secreted by cementocytes and examined their effects on osteoclasts and osteoblasts. Materials and methodsEVs were extracted from the mouse cementocyte cell line, IDG-CM6. The effects of EVs on recombinant RANKL-induced osteoclastogenesis and recombinant Bone morphogenetic protein (BMP)-2-mediated osteoblastogenesis were investigated using the mouse osteoclast progenitor cell line, RAW264.7 and mouse pre-osteoblast cell line, MC3T3-E1, respectively. ResultsEVs enhanced the formation of tartrate-resistant acid phosphatase activity-positive cells. Real-time PCR revealed that EVs up-regulated the expression of osteoclast-related genes. On the other hand, the cell culture supernatant of cementocytes significantly inhibited the differentiation of osteoclasts. Regarding osteoblastogenesis, EVs suppressed the expression of alkaline phosphatase, bone sialoprotein, and osteocalcin induced by recombinant BMP-2 at the gene and protein levels. ConclusionA network of cementocytes, osteoblasts, and osteoclasts may exist in cellular cementum, which suggests the involvement of cementocytes in dynamic metabolism of cementum through EVs.