Stem Cell Pool Research Articles

PLETHORA transcription factors promote early embryo development through induction of meristematic potential.

Plants are dependent on divisions of stem cells to establish cell lineages required for growth. During embryogenesis, early division products are considered to be stem cells, whereas during post-embryonic development, stem cells are present in meristems at the root and shoot apex. PLETHORA/AINTEGUMENTA-LIKE (PLT/AIL) transcription factors are regulators of post-embryonic meristem function and are required to maintain stem cell pools. Despite the parallels between embryonic and post-embryonic stem cells, the role of PLTs during early embryogenesis has not been thoroughly investigated. Here, we demonstrate that the PLT regulome in the zygote, and apical and basal cells is in strong congruence with that of post-embryonic meristematic cells. We reveal that out of all six PLTs, only PLT2 and PLT4/BABY BOOM (BBM) are expressed in the zygote, and that these two factors are essential for progression of embryogenesis beyond the zygote stage and first divisions. Finally, we show that other PLTs can rescue plt2 bbm defects when expressed from the PLT2 and BBM promoters, establishing upstream regulation as a key factor in early embryogenesis. Our data indicate that generic PLT factors facilitate early embryo development in Arabidopsis by induction of meristematic potential.

A dual-color PAX7 and MYF5 in vivo reporter to investigate muscle stem cell heterogeneity in regeneration and aging

Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.

Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle stem cells.

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.

#2145 Sexual dimorphism in kidney regenerative capacity decline and in progressive tubular cell polyploidization during aging

Abstract Background and Aims Aging is characterized by a progressive kidney function decline, resulting in chronic kidney disease (CKD), which is steeper in men than in women. However, sex-related differences in mechanisms underpinning CKD development during aging still remain to be clarified. In most organs regenerative capacity declines overtime from a pool of resident stem cells leading to tissue aging. In the kidney, we have revealed that two adaptive responses co-exist to recover kidney function after injury. On one hand a population of renal progenitors (RPC), which is interspersed among parietal epithelial cells of Bowman's capsule and tubular cells (TC) of nephron segments, proliferate and generate new podocytes and TC to recover structural integrity during the regenerative processes occurring in glomerular and tubular diseases. On the other, TC enter the alternative cell cycles to become polyploid in order to increase their functional capacity and maintain the residual kidney function after injury. However, a continuous TC polyploidization comes with the long term trade-offs (fibrosis and senescence), driving CKD. Here, we aimed to investigate the role of sexual dimorphism in these two different adaptive responses during aging. Method To trace RPC (Pax2+cells) we used the inducible Pax2/Confetti or Pax2/mTmG mice (RPC are randomly labelled by one of the four fluorescent reporter genes or with GFP respectively). To study polyploid TC, we used the inducible Pax8/FUCCI2aR mice (polyploid TC are identified by expression of cell cycle fluorescent proteins in combination with DNA content analysis). Mice were analyzed at 2, 5, 12 and 20 months of age. scRNA sequencing was performed at 2 and 20 months of age. Results Pax2/Confetti mice revealed that the number of Pax2+RPC either of the Bowman's capsule or in tubules was higher in female than in male mice at all ages. Specifically, tracing analysis during aging showed that in females Pax2+RPC of the Bowman's capsule generated new podocytes, resulting in glomeruli with a better integrity of the slit-diaphragm compared with aged males. Moreover, tubular Pax2+RPC in females clonally expanded, generating a higher number of clones, which were longer than those in males. scRNAseq analysis on kidneys showed that females were endowed with a greater number of Pax2+RPC with a higher expression of Pax2 than males, suggesting that RPC in females kept a higher regenerative capacity than males during aging. Simultaneously, analysis of tubular polyploidy demonstrated that, whereas in females the polyploid fraction progressively increased, it was drastically reduced in males during aging. These results suggested that whereas in females the increase of polyploid TC provided a better maintenance of kidney function overtime, loss of polyploid response in males made them more susceptible to develop CKD. Indeed, aged male mice showed a higher percentage of polyploid TC undergoing further cycles of polyploidization, which were associated with fibrosis and senescence. In agreement with these findings, we observed a progressive kidney function decline along with an increase of glomerulosclerosis, interstitial fibrosis and blood pressure, which were significantly greater in males than in females. Conclusion Female mice showed a higher regenerative and polyploid response compared to male mice, aimed likely to cope kidney function decline during aging. These results reveal the intriguing role of a new kidney adaptive response, which may explain the higher susceptibility of males to develop a more premature CKD during aging.

Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications

As caretakers of the hematopoietic system, hematopoietic stem cells assure a lifelong supply of differentiated populations that are responsible for critical bodily functions, including oxygen transport, immunological protection and coagulation. Due to the far-reaching influence of the hematopoietic system, hematological disorders typically have a significant impact on the lives of individuals, even becoming fatal. Hematopoietic cell transplantation was the first effective therapeutic avenue to treat such hematological diseases. Since then, key use and manipulation of hematopoietic stem cells for treatments has been aspired to fully take advantage of such an important cell population. Limited knowledge on hematopoietic stem cell behavior has motivated in-depth research into their biology. Efforts were able to uncover their native environment and characteristics during development and adult stages. Several signaling pathways at a cellular level have been mapped, providing insight into their machinery. Important dynamics of hematopoietic stem cell maintenance were begun to be understood with improved comprehension of their metabolism and progressive aging. These advances have provided a solid platform for the development of innovative strategies for the manipulation of hematopoietic stem cells. Specifically, expansion of the hematopoietic stem cell pool has triggered immense interest, gaining momentum. A wide range of approaches have sprouted, leading to a variety of expansion systems, from simpler small molecule-based strategies to complex biomimetic scaffolds. The recent approval of Omisirge, the first expanded hematopoietic stem and progenitor cell product, whose expansion platform is one of the earliest, is predictive of further successes that might arise soon. In order to guarantee the quality of these ex vivo manipulated cells, robust assays that measure cell function or potency need to be developed. Whether targeting hematopoietic engraftment, immunological differentiation potential or malignancy clearance, hematopoietic stem cells and their derivatives need efficient scaling of their therapeutic potency. In this review, we comprehensively view hematopoietic stem cells as therapeutic assets, going from fundamental to translational.

Asymmetric stem cell division maintains the genetic heterogeneity of tissue cells.

Within a given tissue, the stem cell niche provides the microenvironment for stem cells suitable for their self-renewal. Conceptually, the niche space constrains the size of a stem-cell pool, as the cells sharing the niche compete for its space. It has been suggested that either neutral- or non-neutral-competition of stem cells changes the clone dynamics of stem cells. Theoretically, if the rate of asymmetric division is high, the stem cell competition is limited, thus suppressing clonal expansion. However, the effects of asymmetric division on clone dynamics have never been experimentally tested. Here, using the Drosophila germline stem cell (GSC) system, as a simple model of the in-vivo niche, we examine the effect of division modes (asymmetric or symmetric) on clonal dynamics by combining experimental approaches with mathematical modeling. Our experimental data and computational model both suggest that the rate of asymmetric division is proportional to the time a stem cell clone takes to expand. Taken together, our data suggests that asymmetric division is essential for maintaining the genetic variation of stem cells and thus serves as a critical mechanism for safeguarding fertility over the animal age or preventing multiple disorders caused by the clonal expansion of stem cells.

Regulatory Role of Meox1 in Muscle Growth of Sebastes schlegelii.

Meox1 is a critical transcription factor that plays a pivotal role in embryogenesis and muscle development. It has been established as a marker gene for growth-specific muscle stem cells in zebrafish. In this study, we identified the SsMeox1 gene in a large teleost fish, Sebastes schlegelii. Through in situ hybridization and histological analysis, we discovered that SsMeox1 can be employed as a specific marker of growth-specific muscle stem cells, which originate from the somite stage and are primarily situated in the external cell layer (ECL) and myosepta, with a minor population distributed among muscle fibers. The knockdown of SsMeox1 resulted in a significant increase in Ccnb1 expression, subsequently promoting cell cycle progression and potentially accelerating the depletion of the stem cell pool, which ultimately led to significant growth retardation. These findings suggest that SsMeox1 arrests the cell cycle of growth-specific muscle stem cells in the G2 phase by suppressing Ccnb1 expression, which is essential for maintaining the stability of the growth-specific muscle stem cell pool. Our study provides significant insights into the molecular mechanisms underlying the indeterminate growth of large teleosts.

DDX20 is required for cell-cycle reentry of prospermatogonia and establishment of spermatogonial stem cell pool during testicular development in mice

RNA-binding proteins (RBPs), as key regulators of mRNA fate, are abundantly expressed in the testis. However, RBPs associated with human male infertility remain largely unknown. Through bioinformatic analyses, we identified 62 such RBPs, including an evolutionarily conserved RBP, DEAD-box helicase 20 (DDX20). Male germ-cell-specific inactivation of Ddx20 at E15.5 caused T1-propsermatogonia (T1-ProSG) to fail to reenter cell cycle during the first week of testicular development in mice. Consequently, neither the foundational spermatogonial stem cell (SSC) pool nor progenitor spermatogonia were ever formed in the knockout testes. Mechanistically, DDX20 functions to control the translation of its target mRNAs, many of which encode cell-cycle-related regulators, by interacting with key components of the translational machinery in prospermatogonia. Our data demonstrate a previously unreported function of DDX20 as a translational regulator of critical cell-cycle-related genes, which is essential for cell-cycle reentry of T1-ProSG and formation of the SSC pool.

Associations of Early-Life and Adult Anthropometric Measures with the Expression of Stem Cell Markers CD44, CD24, and ALDH1A1 in Women with Benign Breast Biopsies.

According to the stem cell hypothesis, breast carcinogenesis may be related to the breast stem cell pool size. However, little is known about associations of breast cancer risk factors, such as anthropometric measures, with the expression of stem cell markers in noncancerous breast tissue. The analysis included 414 women with biopsy-confirmed benign breast disease in the Nurses' Health Study and Nurses' Health Study II. Birthweight, weight at age 18, current weight, and current height were reported via self-administered questionnaires. IHC staining of stem cell markers (CD44, CD24, and aldehyde dehydrogenase family 1 member A1) in histopathologically normal epithelial and stromal breast tissue was quantified using an automated computational image analysis system. Linear regression was used to examine the associations of early-life and adult anthropometric measures with log-transformed stem cell marker expression, adjusting for potential confounders. Birthweight [≥10.0 vs. <5.5 lbs: β (95% confidence interval) = 4.29 (1.02, 7.56); P trend = 0.001 in the stroma] and adult height [≥67.0 vs. <63.0 inch: 0.86 (0.14, 1.58); P trend = 0.02 in the epithelium and stroma combined] were positively associated with CD44 expression. Childhood body fatness was inversely associated (P trend = 0.03) whereas adult height was positively associated with CD24 expression in combined stroma and epithelium (P trend = 0.03). Our data suggest that anthropometric measures, such as birthweight, adult height, and childhood body fatness, may be associated with the stem cell expression among women with benign breast disease. Anthropometric measures, such as birthweight, height, and childhood body fatness, may have long-term impacts on stem cell population in the breast.

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

Environmentally relevant concentrations of benzophenones exposure disrupt intestinal homeostasis, impair the intestinal barrier, and induce inflammation in mice

The aim of this study is to investigate the adverse effects of benzophenones (BPs) on the intestinal tract of mice and the potential mechanism. F1-generation ICR mice were exposed to BPs (benzophenone-1, benzophenone-2, and benzophenone-3) by breastfeeding from birth until weaning, and by drinking water after weaning until maturity. The offspring mice were executed on postnatal day 56, then their distal colons were sampled. AB-PAS staining, HE staining, immunofluorescence, Transmission Electron Microscope, immunohistochemistry, Western Blot and RT-qPCR were used to study the effects of BPs exposure on the colonic tissues of offspring mice. The results showed that colonic microvilli appeared significantly deficient in the high-dose group, and the expression of tight junction markers Zo-1 and Occludin was significantly down-regulated and the number of goblet cells and secretions were reduced in all dose groups, and the expression of secretory cell markers MUC2 and KI67 were decreased, as well as the expression of intestinal stem cell markers Lgr5 and Bmi1, suggesting that BPs exposure caused disruption of intestinal barrier and imbalance in the composition of the intestinal stem cell pool. Besides, the expression of cellular inflammatory factors such as macrophage marker F4/80 and tumor necrosis factor TNF-α was elevated in the colonic tissues of all dose groups, and the inflammatory infiltration was observed, which means the exposure of BPs caused inflammatory effects in the intestinal tract of F1-generation mice. In addition, the contents of Notch/Wnt signaling pathway-related genes, such as Dll-4, Notch1, Hes1, Ctnnb1and Sfrp2 were significantly decreased in each high-dose group (P < 0.05), suggesting that BPs may inhibit the regulation of Notch/Wnt signaling pathway. In conclusion, exposure to BPs was able to imbalance colonic homeostasis, disrupt the intestinal barrier, and trigger inflammation in the offspring mice, which might be realized through interfering with the Notch/Wnt signaling pathway.

Extracellular Caspase-1 induces hair stem cell migration in wounded and inflamed skin conditions.

The wound-healing process is a paradigm of the directed migration of various pools of stem cells from their niche to the site of injury where they replenish damaged cells. Two decades have elapsed since the observation that wounding activates multipotent hair follicle stem cells to infiltrate the epidermis, but the cues that coax these cells out of their niche remain unknown. Here, we report that Caspase-1, a protein classically known as an integral component of the cytosolic inflammasome, is secreted upon wounding and has a non-canonical role in the extracellular milieu. Through its caspase activation recruitment domain (CARD), Caspase-1 is sufficient to initiate the migration of hair follicle stem cells into the epidermis. Uncovering this novel function of Caspase-1 also facilitates a deeper understanding of the mechanistic basis of the epithelial hyperplasia found to accompany numerous inflammatory skin diseases.

ASH2L Mediates Epidermal Differentiation and Hair Follicle Morphogenesis through H3K4me3 Modification

The processes of epidermal development in mammals are regulated by complex molecular mechanisms, such as histone modifications. Histone H3 lysine K4 methylation mediated by COMPASS (complex of proteins associated with Set1) methyltransferase is associated with gene activation, but its effect on epidermal lineage development remains unclear. Therefore, we constructed a mouse model of specific ASH2L (COMPASS methyltransferase core subunit) deletion in epidermal progenitor cells and investigated its effect on the development of mouse epidermal lineage. Furthermore, downstream target genes regulated by H3K4me3 were screened using RNA sequencing combined with Cleavage Under Targets and Tagmentation sequencing. Deletion of ASH2L in epidermal progenitor cells caused thinning of the suprabasal layer of the epidermis and delayed hair follicle morphogenesis in newborn mice. These phenotypes may be related to the reduced proliferative capacity of epidermal and hair follicle progenitor cells. ASH2L depletion may also lead to depletion of the epidermal stem cell pools in late mouse development. Finally, genes related to hair follicle development (Shh, Edar, and Fzd6), Notch signaling pathway (Notch2, Notch3, Hes5, and Nrarp), and ΔNp63 were identified as downstream target genes regulated by H3K4me3. Collectively, ASH2L-dependent H3K4me3 modification served as an upstream epigenetic regulator in epidermal differentiation and hair follicle morphogenesis in mice.

182 Purification and Characterization of Human Neural Stem and Progenitor Cells

INTRODUCTION: The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. METHODS: We combine high-dimensional flow cytometry with single cell transcriptomics to comprehensively purify and functionally characterize NSPCs from the developing human brain during mid-gestation. We utilize index sorting based on the combinatorial expression of cell-surface markers (immunophenotype) to directly link surface-marker expression to the expressed transcriptomes of single cells, allowing for rigorous validation of sort purity. We further demonstrate the functional properties of each identified cell type in vitro and in vivo. RESULTS: CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain.THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+ THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a novel transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell, which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. CONCLUSIONS: The results presented here are strong proof-of-concept that distinct cell types from the developing brain can be prospectively isolated based on the expression of multiple surface markers. The combined results from scRNA-seq, in vitro differentiation, and in vivo transplantation offers the most rigorous interrogation of NSPCs’ transcriptomic identities and functional behavior. The modular nature of our antibody panel allows for the easy expansion or simplification of the panel, based on the cell-type of interest, thus providing a valuable tool for future investigation of both molecular and functional NSPC heterogeneity.

Following the p63/Keratin5 basal cells in the sensory and non-sensory epithelia of the vomeronasal organ.

The vomeronasal organ (VNO) is a part of the accessory olfactory system, which detects pheromones and chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares several features with the epithelium of the main olfactory epithelium (MOE). However, it is a distinct neuroepithelium populated by chemosensory neurons that differ from the olfactory sensory neurons in cellular structure, receptor expression, and connectivity. The vomeronasal organ of rodents comprises a sensory epithelium (SE) and a thin non-sensory epithelium (NSE) that morphologically resembles the respiratory epithelium. Sox2-positive cells have been previously identified as the stem cell population that gives rise to neuronal progenitors in MOE and VNE. In addition, the MOE also comprises p63 positive horizontal basal cells, a second pool of quiescent stem cells that become active in response to injury. Immunolabeling against the transcription factor p63, Keratin-5 (Krt5), Krt14, NrCAM, and Krt5Cre tracing experiments highlighted the existence of horizontal basal cells distributed along the basal lamina of SE of the VNO. Single cell sequencing and genetic lineage tracing suggest that the vomeronasal horizontal basal cells arise from basal progenitors at the boundary between the SE and NSE proximal to the marginal zones. Moreover, our experiments revealed that the NSE of rodents is, like the respiratory epithelium, a stratified epithelium where the p63/Krt5+ basal progenitor cells self-replicate and give rise to the apical columnar cells facing the lumen of the VNO.

HnRNPU is required for spermatogonial stem cell pool establishment in mice

The continuous regeneration of spermatogonial stem cells (SSCs) underpins spermatogenesis and lifelong male fertility, but the developmental origins of the SSC pool remain unclear. Here, we document that hnRNPU is essential for establishing the SSC pool. In male mice, conditional loss of hnRNPU in prospermatogonia (ProSG) arrests spermatogenesis and results in sterility. hnRNPU-deficient ProSG fails to differentiate and migrate to the basement membrane to establish SSC pool in infancy. Moreover, hnRNPU deletion leads to the accumulation of ProSG and disrupts the process of T1-ProSG to T2-ProSG transition. Single-cell transcriptional analyses reveal that germ cells are in a mitotically quiescent state and lose their unique identity upon hnRNPU depletion. We further show that hnRNPU could bind to Vrk1, Slx4, and Dazl transcripts that have been identified to suffer aberrant alternative splicing in hnRNPU-deficient testes. These observations offer important insights into SSC pool establishment and may have translational implications for male fertility.

Homeostatic regulation of renewing tissue cell populations via crowding control: stability, robustness and quasi-dedifferentiation

To maintain renewing epithelial tissues in a healthy, homeostatic state, cell divisions and differentiation need to be tightly regulated. Mechanisms of homeostatic regulation often rely on crowding feedback control: cells are able to sense the cell density in their environment, via various molecular and mechanosensing pathways, and respond by adjusting division, differentiation, and cell state transitions appropriately. Here, we determine, via a mathematically rigorous framework, which general conditions for the crowding feedback regulation (i) must be minimally met, and (ii) are sufficient, to allow the maintenance of homeostasis in renewing tissues. We show that those conditions naturally allow for a degree of robustness toward disruption of regulation. Furthermore, intrinsic to this feedback regulation is that stem cell identity is established collectively by the cell population, not by individual cells, which implies the possibility of ‘quasi-dedifferentiation’, in which cells committed to differentiation may reacquire stem cell properties upon depletion of the stem cell pool. These findings can guide future experimental campaigns to identify specific crowding feedback mechanisms.

Abstract 1259: HMGA1: An epigenetic gatekeeper of Wnt signals during colon tumorigenesis and regeneration

Abstract Cancer cells undergo epigenetic reprogramming to co-opt stem cell networks and drive tumor progression, although how this occurs remains unclear. The High Mobility Group A1 (HMGA1) gene encodes an architectural transcription factor that binds to DNA at AT-rich sequences where it recruits chromatin complexes to modulate gene expression. HMGA1 is highly expressed during embryogenesis and in adult stem cells, but silenced in differentiated cells. HMGA1 becomes re-expressed in aggressive cancers where high levels portend adverse outcomes. In colorectal cancer (CRC), HMGA1 is among the genes most highly overexpressed compared to normal colon epithelium. We previously discovered that HMGA1 is required for oncogenic phenotypes in CRC cell lines by inducing genes involved in an epithelial-mesenchymal transition. Hmga1 transgenic mice also develop aberrant proliferation and polyposis of small intestinal epithelium where Hmga1 amplifies Wnt signals to enhance self-renewal. To determine how HMGA1 functions in colon epithelial homeostasis and tumorigenesis, we examined mouse models with overexpression or deficiency of Hmga1. Here, we uncover a previously unknown role for HMGA1 as an epigenetic gatekeeper of Wnt signals in colon epithelial cells, both during tumorigenesis driven by mutant APC or stress-induced tissue repair. Strikingly, loss of just a single Hmga1 allele within the colon epithelium disrupts tumorigenesis while prolonging survival in two models of colon tumorigenesis driven by APC mutation, including CDX2P-CreERTCApcfl/fl and APCMin mice inoculated with pro-carcinogenic, enterotoxigenic Bacteroides fragilis. Single cell RNA sequencing in Apc mutant crypt epithelial cells reveals that intact Hmga1 is required for expansion in the Lgr5+ stem cell pool, deep secretory cells, and tuft cells during tumorigenesis. Hmga1 deficiency impacts the stem cell pool the greatest, decreasing Lgr5+ stem cells with skewing to more proliferative, transit amplifying-like cells. Single cell transcriptomic analyses together with assays of single cell chromatin accessibility demonstrate that Hmga1 maintains accessible chromatin to activate gene loci involved in Wnt signals, including Ascl2, a stem cell master regulator, Wnt agonist receptors, and Wnt downstream effectors. Surprisingly, either depletion or overexpression of Hmga1 has no impact on colon epithelial regeneration under homeostatic conditions, although Hmga1 overexpression enhances tissue repair and survival following injury induced by inflammation or irradiation. Together, our results establish HMGA1 as an epigenetic gatekeeper of Wnt signals in colon stem cells during APC-driven tumorigenesis or injury, but not steady state homeostasis, highlighting its role as a promising therapeutic target for both cancer and regenerative medicine. Citation Format: Linda M. S. Resar, Li Z. Luo, Lingling Xian, Iliana Herrera, Shaoguang Wu, Jung-Hyun Kim, Ying Feng, Julian Calderon-Espinosa, Eric R. Fearon, Cynthia Sears. HMGA1: An epigenetic gatekeeper of Wnt signals during colon tumorigenesis and regeneration [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 1259.

Abstract 1896: Therapeutic potential of TORL-4-500, an antibody-drug conjugate directed against delta like non-canonical notch ligand 1 (DLK1)

Abstract Delta like non-canonical Notch ligand 1 (DLK1) is a transmembrane protein that belongs to the NOTCH non-canonical ligand family. It has been implicated in adipogenesis, the regulation of stem cell pools, tissue differentiation during development, cancer differentiation, and cancer stem-like cell maintenance. DLK1 is highly expressed in adrenocortical, uterine, and testicular cancers as well as in a large portion of pancreatic, sarcoma, liver and squamous lung cancer patient samples. In contrast there is limited normal tissue expression beyond the normal adrenal gland, pituitary, and ovarian tissue samples. This expression profile makes DLK1 an attractive target for development of a therapeutic antibody-drug conjugate (ADC). This study describes the generation and preclinical characterization of TORL-4-500, an ADC consisting of a humanized anti-DLK1 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker. TORL-4-500 showed strong binding to DLK1 by flow cytometry in DLK1 native and artificial overexpressing cell lines. In contrast no binding was observed in DLK1 non-expressing cell lines. TORL-4-500 exhibits nanomolar binding affinity for both human and cynomolgus monkey DLK1 and is rapidly internalized in DLK1 expressing cells. TORL-4-500 exhibited selective efficacy in cell line xenograft models of DLK1 positive human cancers. Treatment with TORL-4-500 induced significant regressions in four DLK1 expressing human cancer cell line xenograft studies encompassing liver, small cell lung cancer (SCLC) and sarcoma cancer. Furthermore, anti-tumor responses were sustained in each of the DLK1 expressing models for several weeks post-final dose. In the case of the two SCLC cell lines, complete regressions of xenograft tumors were measured out past 100 days in the TORL-4-500 treated animals. No significant impact on xenograft tumor growth was observed in either a DLK1 non-expressing colon cell line xenograft or in a DLK1 non-expressing melanoma xenograft study. Each of the doses tested in this study was well tolerated in mice with no dose-limiting toxicity observed. The nonclinical pharmacokinetics and toxicokinetics of TORL-4-500 were characterized in mice and monkeys and results support dosing in humans. In summary, TORL-4-500 is a novel therapeutic for DLK1 positive cancers and on the basis of these promising preclinical efficacy results, a first in human trial to evaluate safety, tolerability, pharmacokinetics, and antitumor activity of TORL-4-500 has been launched in patients with advanced cancer and is currently ongoing (NCT06005740). Citation Format: Martina S. McDermott, Neil A. O'Brien, Ming Lu, Jun Zhang, KeWei Gong, Benjamin Hoffstrom, Tong Luo, Min Liang, Weiping Jia, Kevin Chau, Leonard Presta, John Glaspy, Dennis J. Slamon. Therapeutic potential of TORL-4-500, an antibody-drug conjugate directed against delta like non-canonical notch ligand 1 (DLK1) [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 1896.