Cell Hierarchy Research Articles

Lgr5 and stem/progenitor gene expression in gastric/gastroesophageal junction carcinoma \u2013 significance of potentially retained stemness

BackgroundGastric/gastroesophageal junction (GEJ) adenocarcinomas are heterogeneous, comprising four molecularly distinct subtypes, namely EBV-positive, microsatellite instability (MSI), chromosomal instability (CIN) and genomically stable (GS) subtypes, and a part of this heterogeneity may hypothesized to be different cells-of-origin. Stem/progenitor cell hierarchy in the stomach is complex, which include the Lgr5(+) gastric stem cells (GSCs).MethodsWhile previous studies have focused on non-nuclear Lgr5 expression, nuclear Lgr5 expression has been reported in a subset of stem cells, and we examined nuclear Lgr5 expression in a local cohort of 95 cases of gastric/GEJ adenocarcinoma. mRNA levels for LGR5 and other stem cell marker genes were examined in the TCGA cohort.ResultsWe observed nuclear Lgr5 expression in a 18/95 cases. Near mutual exclusivity was seen between nuclear Lgr5 and strong non-nuclear Lgr5. Both strong non-nuclear and nuclear Lgr5 expression tended to be seen more frequently with the intestinal histotype and approximated CIN molecular subtype. With respect to overall survival (OS), nuclear Lgr5 expression appears to be protective, with the worst survival being seen in the cases lacking nuclear Lgr5 and with low non-nuclear Lgr5 expression. When compared to other stem/progenitor cell markers, LGR5 mRNA expression clusters with other GSC marker genes, including VIL1. Higher expression of these GSC marker genes was associated with better OS.ConclusionsOur results show that Lgr5 expression is dynamic in gastric/GEJ adenocarcinoma and heterogeneous across the several disease attributes. We postulate that this may reflect “retained stemness” in the form of Lgr5High-GSC signature that appears to be associated with better survival.

The combination of dibenzazepine and a DOT1L inhibitor enables a stable maintenance of human naïve-state pluripotency in non-hypoxic conditions.

Conventional human pluripotent stem cells (hPSCs), known for being in a primed state, are pivotal for both basic research and clinical applications since such cells produce various types of differentiated cells. Recent reports on PSCs shed light on the pluripotent hierarchy of stem cells and have promoted the exploration of new stem cell states along with their culture systems. Human naïve PSCs are expected to provide further knowledge of early developmental mechanisms and improvements for differentiation programmes in the regenerative therapy of conventionally primed PSCs. However, practical challenges exist in using naïve-state PSCs such as determining the conditions for hypoxic culture condition and showing limited stable cellular proliferation. Here, we have developed new leukemia inhibitory factor dependent PSCs by applying our previous work, the combination of dibenzazepine and a DOT1L inhibitor to achieve the stable culture of naïve-state PSCs. The potential of these cells to differentiate into all three germ layers was shown both in vitro and in vivo. Such new naïve-state PSCs formed dome-shaped colonies at a faster rate than conventional, primed-state human induced PSCs and could be maintained for an extended period in the absence of hypoxic culture conditions. We also identified relatively high expression levels of naïve cell markers. Thus, non-hypoxia treated, leukemia inhibitory factor-dependent PSCs are anticipated to have characteristics similar to those of naïve-like PSCs, and to enhance the utility value of PSCs. Such naïve PSCs may allow the molecular characterization of previously undefined naïve human PSCs, and to ultimately contribute to the use of human pluripotent stem cells in regenerative medicine and disease modelling.

Defined Mathematical Relationships Among Cancer Cells Suggest Modular Growth in Tumor Progression and Highlight Developmental Features Consistent With a Para-Embryonic Nature of Cancer.

Several similarities between the embryo development and the cancer process suggest the para-embryonic nature of tumors. Starting from an initial cancer stem cell (i-CSC) as a para-embryonic stem cell (p-ESC), a hierarchic sequence of CSCs (CSC1s, CSC2s, CSC3s) and non-CSCs [cancer progenitor cells (CPCs), cancer differentiated cells (CDCs)] would be generated, mimicking an ectopic rudimentary ontogenesis. Such a proposed heterogeneous cell hierarchy within the tumor structure would suggest a tumor growth model consistent with experimental data reported for mammary tumors. By tabulating the theoretical data according to this model, it is possible to identify defined mathematical relationships between cancer cells (CSCs and non-CSCs) that are surprisingly similar to experimental data. Moreover, starting from this model, it is possible to speculate that, during progression, tumor growth would occur in a modular way that recalls the propagation of tumor spheres in vitro. All these considerations favor a comparison among normal blastocysts (as in vitro embryos), initial avascular tumors (as in vivo abnormal blastocysts) and tumor spheres (as in vitro abnormal blastocysts). In conclusion, this work provides further support for the para-embryonic nature of the cancer process, as recently theorized.

Whole transcriptome analysis of bovine mammary progenitor cells by P-Cadherin enrichment as a marker in the mammary cell hierarchy

Adult bovine mammary stem cells possess the ability to regenerate in vivo clonal outgrowths that mimic functional alveoli. Commonly available techniques that involve immunophenotype-based cell sorting yield cell fractions that are moderately enriched, far from being highly purified. Primary bovine mammary epithelial cells segregated in four different populations according to the expression of P-Cadherin and CD49f. Sorted cells from each fraction were tested for the presence of lineage-restricted progenitors and stem cells. Only cells from the CD49fhigh/P-Cadherinneg subpopulation were able to give rise to both luminal- and myoepithelial-restricted colonies in vitro and generate organized outgrowths in vivo, which are hallmarks of stem cell activity. After whole transcriptome analysis, we found gene clusters to be differentially enriched that relate to cell-to-cell communication, metabolic processes, proliferation, migration and morphogenesis. When we analyzed only the genes that were differentially expressed in the stem cell enriched fraction, clusters of downregulated genes were related to proliferation, while among the upregulated expression, cluster of genes related to cell adhesion, migration and cytoskeleton organization were observed. Our results show that P-Cadherin separates mammary subpopulations differentially in progenitor cells or mammary stem cells. Further we provide a comprehensive observation of the gene expression differences among these cell populations which reinforces the assumption that bovine mammary stem cells are typically quiescent.

Abstract 5724: Revelation of shared transcriptional gradients in glioblastoma tumors and cultured glioma stem cells

Abstract The brain tumor Glioblastoma (GBM) is a virtual death sentence for anyone who is diagnosed, with a median survival time of 12-15 months with standard treatments1 and a 5 year survival rate of under 10% 2. There is a strong body of evidence supporting the existence of stem like cells, termed glioma stem cells (GSCs), that can repopulate the tumor after removal and therapy application3-6. Thus, GSCs present a tantalizing target for potential therapies against GBM. However, studies of GSCs have shown heterogeneity at the level of the transcriptome and drug response5,7, suggesting that there is significant biological variation that translates into differential sensitivity to various drugs. A full characterization of biological heterogeneity may aid in the search for targeted therapies. Here, we profile the transcriptomes of 72 patient derived GSC cultures, and obtain scRNA-seq on 29 cultures from 26 patients (> 69,000 cells) plus 5 GBM tumors (> 14,000 cells). With this data, we find two anticorrelated transcriptional programs in the GSC cultures, one associated with immune or injury response related pathways and the other with neural developmental pathways. We then compare the GSC cultures to patient tumors in the scRNA-seq data, and find that a portion of GBM tumor cells are similar to GSC cultures. We find that a gradient between GSC and astrocyte programs separates cells with stemness properties from those that are not stem-like, and that within stem-like tumor cells and non stem-like tumor cells a gradient between the neural developmental and immune related programs exists as was seen for the cultured GSCs. In GSCs, we also find epigenetic variation in DNA methylation associated with the developmental and immune related programs. Overall these data suggest variation between two biological programs manifests at the level of gene expression and epigenetic regulation in GSCs in tumors. 1. Stupp, R. et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 352, 987-996 (2005). 2. Brennan, C. W. et al. The somatic genomic landscape of glioblastoma. Cell 155, 462-477 (2013). 3. Singh, S. K. et al. Identification of human brain tumour initiating cells. Nature 432, 396-401 (2004). 4. Chen, J. et al. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature 488, 522-526 (2012). 5. Lan, X. et al. Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy. Nature 549, 227-232 (2017). 6. Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396-1401 (2014). 7. Meyer, M. et al. Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity. Proc. Natl. Acad. Sci. U. S. A. 112, 851-856 (2015). Citation Format: Owen K. Whitley, Laura M. Richards, Florence Cavalli, Paul Guilhamon, Fiona Coutinho, Michelle Kushida, H. Artee Luchman, Samuel Weiss, Mathieu Lupien, Peter Dirks, Trevor Pugh, Gary Bader. Revelation of shared transcriptional gradients in glioblastoma tumors and cultured glioma stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5724.

2007 – DIFFERENTIAL ALTERNATIVE POLYADENYLATION LANDSCAPES MEDIATE HEMATOPOIETIC STEM CELL ACTIVATION AND REGULATE GLUTAMINE METABOLISM

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3'-untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies in vivo remains elusive. Using extensive in vitro and in vivo analysis approaches we show that deregulation of the APA regulator Pabpn1 results in severe hematopoietic stem cell (HSC) defects. Further, we performed low input 3'-sequencing and established bioinformatic pipelines to uncover dynamic APA patterns in numerous genes of HSCs and multipotent progenitors determining the genome-wide APA landscape (APAome). This revealed transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation (Sommerkamp et al., Cell Stem Cell, 2020). Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching. This process is mediated by Nudt21 and is required for the proper stress response of HSCs. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Inhibition of this metabolic adjustment leads to impaired HSC function and a partial block in differentiation. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal and commitment.

Abstract IA31: Stalled developmental programs at the root of K27M mutant gliomas and other pediatric brain tumors

Abstract Childhood brain tumors have suspected prenatal origins. We showed that H3K27M in gliomas impairs the production and the spread of the repressive H3K27me3 mark from PRC2 high-affinity sites. Neither the recruitment of PRC2 to its nucleation sites nor the deposition of the H3K27me3 mark in the proximity of those sites was affected by the mutation. However, our findings indicate that as the marks cannot spread to establish the proper silencing landscape, further lineage specification, a major role of PRC2, is not possible, and the cell is stalled in an early epigenetic and progenitor state, indefinitely multiplying without being able to further differentiate. To identify vulnerable developmental states, we generated a single-cell transcriptome atlas of >44,000 cells from embryonal pons and forebrain, two major tumor locations. We derived signatures for 119 distinct cell populations and defined regional cellular diversity and differentiation dynamics. Projection of bulk tumor transcriptomes onto this dataset shows that WNT medulloblastomas match the rhombic lip-derived mossy fiber neuronal lineage, embryonal tumors with multilayered rosettes fully recapitulate a cortical neuronal lineage, atypical teratoid-rhabdoid tumors originate outside of the neuro-ectoderm, while diffuse intrinsic pontine gliomas resemble a pontine astrocytic progenitor and differentiate upon removal of driver mutation H3K27M. Importantly, single-cell tumor profiles reveal highly defined cell hierarchies mirroring transcriptional programs of the corresponding normal lineages. We identify impaired differentiation of specific neural progenitors as a common mechanism underlying these pediatric cancers and provide a rational framework for future modeling and therapeutic interventions. Citation Format: Nada Jabado. Stalled developmental programs at the root of K27M mutant gliomas and other pediatric brain tumors [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA31.

Design and Mechanical Testing of 3D Printed Hierarchical Lattices Using Biocompatible Stereolithography

Emerging 3D printing technologies are enabling the rapid fabrication of complex designs with favorable properties such as mechanically efficient lattices for biomedical applications. However, there is a lack of biocompatible materials suitable for printing complex lattices constructed from beam-based unit cells. Here, we investigate the design and mechanics of biocompatible lattices fabricated with cost-effective stereolithography. Mechanical testing experiments include material characterization, lattices rescaled with differing unit cell numbers, topology alterations, and hierarchy. Lattices were consistently printed with 5% to 10% lower porosity than intended. Elastic moduli for 70% porous body-centered cube topologies ranged from 360 MPa to 135 MPa, with lattices having decreased elastic moduli as unit cell number increased. Elastic moduli ranged from 101 MPa to 260 MPa based on unit cell topology, with increased elastic moduli when a greater proportion of beams were aligned with the loading direction. Hierarchy provided large pores for improved nutrient transport and minimally decreased lattice elastic moduli for a fabricated tissue scaffold lattice with 7.72 kN/mm stiffness that is suitable for bone fusion. Results demonstrate the mechanical feasibility of biocompatible stereolithography and provide a basis for future investigations of lattice building blocks for diverse 3D printed designs.

Colon Cancer Heterogeneity: Welcome to the RiboZone.

In this issue of Cell Stem Cell, Morral etal. (2020) shed new light on the hierarchical organization of cells within colorectal cancer. They show that tumor cells at the apex of this hierarchy reside in particular tumor zones and possess high protein synthesis activity. Interference with their biosynthetic activity results in an irreversible growth arrest of CRC.

Zones of biosynthesis.

Morral, Stanisavljevic et al. show that colorectal cancer (CRC) cell hierarchy is based on the capacity to perform biosynthesis. Undifferentiated CRC cells that have high capacity for protein synthesis were found immediately adjacent to the stroma, and this capacity was lost upon differentiation.

Zonation of Ribosomal DNA Transcription Defines a Stem Cell Hierarchy in Colorectal Cancer.

Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here, we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occurs in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA polymerase I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5+ and LGR5- tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins.

OR03-02 Identification of a Novel Stem/Progenitor Population of the Adrenal Medulla

The adrenal glands regulate multiple physiological processes including the stress response, the immune system and metabolism. The adrenal is composed of an outer cortex that produces steroids, and an inner medulla that produces catecholamines. Tissue-specific stem/progenitor populations have been identified in the adrenal cortex, while the presence of a functional stem/progenitor population in the adrenal medulla is unclear. The adrenal medulla derives from the neural crest and contains chromaffin cells, neurons and sustentacular (support) cells. Establishing cell hierarchy and elucidating mechanisms of regulation of the different cell types is important to understand normal homeostasis and disease pathogenesis, such as of pheochromocytomas. Using genetic approaches in mouse, we have established that a subpopulation of sustentacular cells express the stem/progenitor marker SOX2. Through genetic lineage-tracing using the Sox2-CreERT2 strain, we demonstrate that these are an expanding population, capable of giving rise to chromaffin cells and neurons throughout life, consistent with a stem/progenitor role in vivo. We further demonstrate the self-renewal and differentiation potential of SOX2+ cells through in vitro isolation and expansion. Through analysis of FFPE sections of human adrenals, we confirm the presence of SOX2+ cells in the normal adult organ, as well as in pheochromocytomas. Taken together, our data support the identification of a previously undescribed stem/progenitor cell in the mammalian adrenal medulla, and confirm its functional relevance.

Maturation State of HIV Infected CD4 T Cells Impacts the Inducible Response of the Latent Viral Reservoir

Abstract A structured microenvironment is necessary for proper immune cell function. While it is rarely possible to use ex vivo experimental models of human tissue (e.g. tonsil), culture of multiple immune cell types together in close contact may benefit cell survival and function. To study HIV latency in primary CD4 T cells, we use an in vitro model, which produces an infected cell hierarchy closely resembling that found in vivo: HIV frequency in effector memory (TEM) > central memory (TCM) > naïve (TN) cells. To enable mechanistic investigations, we developed a method to track CD4 cell phenotype origins following long-term culture and T cell receptor (TCR) stimulation. TN, TCM and TEM subsets were isolated by sorting and stained with live-cell tracking dyes: carboxyfluorescein (CFSE), Yellow and Violet dye, respectively. The tagged subsets were pooled with unstained CD4 cells to help preserve a healthy microenvironment. This mixture was co-cultured with autologous productively infected, TCR-activated cells to facilitate cell-to-cell HIV transmission and establishment of latency. After 3 days, dye-labeled cells were removed by sorting and rested an additional 3 days to complete provirus integration. Latent HIV reactivation by TCR stimulation was measured with HIV RNA expression by PrimeFlow analysis. Residual dye staining provided inference of initial subset origin on a single cell basis. Using this design improved viability and survival for all maturation subsets, compared to isolating subsets prior to stimulation. Results showed that HIV provirus in TN cells was the most responsive to TCR activation. In addition, our study highlights a novel approach that can easily be adapted to other rare cell subsets in biologically relevant settings.

Identification of very small cancer stem cells expressing hallmarks of pluripotency in B16F10 melanoma cells and their reoccurrence in B16F10-derived clones

Melanoma is characterized by high heterogeneity and plasticity, most likely due to the presence of mutated melanocyte stem cells or immature progenitor cells in the skin that serves as precursors to melanoma. In the present study, for the first time, we identified rare cells in the murine melanoma B16F10, and human A2058 and SK-MEL-28 cell lines that express pluripotency markers, including Oct4, Nanog, Sox2 and a marker of melanoma cancer cells (ALDH1/2). These cells are very small with round morphology and they grow onto melanoma cells, thereby demonstrating feeder layer dependence similar to that of other pluripotent cells. These cells underwent self-renewal, symmetric and asymmetric division. We called these cells murine very small cancer stem cells (VSCSC). VSCSC were also found in B16F10-derived clones after 3–5 consecutive passages, where they occur as single cells or as small colonies, nevertheless, always using melanoma cells as feeders. These cells formed melanospheres enriched with Oct4-and ALDH1/2-positive cells. We also evaluated the possible effect of VSCSC that presented in the parental cell line (B16F10) and in clones based on their functional characteristics. We found that VCSCS present in the B16F10 cell line reappearing in their clones were required for continuous tumor growth and were responsible for melanoma cell heterogeneity and plasticity rather than directly affecting functional characteristics of melanoma cells. Our data, together with those of previous reports suggested the existence of melanoma-competent melanocyte stem cells, which corroborate the hypothesis of the existence of tumor-initiating cells and cancer stem cell hierarchies, at least in melanoma.

A unified simulation model for understanding the diversity of cancer evolution.

Because cancer evolution underlies the therapeutic difficulties of cancer, it is clinically important to understand the evolutionary dynamics of cancer. Thus far, a number of evolutionary processes have been proposed to be working in cancer evolution. However, there exists no simulation model that can describe the different evolutionary processes in a unified manner. In this study, we constructed a unified simulation model for describing the different evolutionary processes and performed sensitivity analysis on the model to determine the conditions in which cancer growth is driven by each of the different evolutionary processes. Our sensitivity analysis has successfully provided a series of novel insights into the evolutionary dynamics of cancer. For example, we found that, while a high neutral mutation rate shapes neutral intratumor heterogeneity (ITH) characterized by a fractal-like pattern, a stem cell hierarchy can also contribute to shaping neutral ITH by apparently increasing the mutation rate. Although It has been reported that the evolutionary principle shaping ITH shifts from selection to accumulation of neutral mutations during colorectal tumorigenesis, our simulation revealed the possibility that this evolutionary shift is triggered by drastic evolutionary events that occur in a short time and confer a marked fitness increase on one or a few cells. This result helps us understand that each process works not separately but simultaneously and continuously as a series of phases of cancer evolution. Collectively, this study serves as a basis to understand in greater depth the diversity of cancer evolution.

Reprogramming cell fates by RNA binding proteins in stem cells and cancer

Stem cells and cancer share a characteristic potential of self‐renewal, which is essential for long‐term maintenance of both cell types in vivo. Similar to a hierarchy of stem and progenitor cells in normal tissues, many types of tumors are maintained by a small population of self‐renewing cancer cells while the bulk of the tumor is consisted of more differentiated cells with no or limited renewal activity. The self‐renewing cancer cells, or cancer stem cells, are often resistant to conventional therapies and thereby mediate tumor relapse. With the ability to self‐renew and propagate the tumor, cancer stem cells also contribute to metastasis and disease progression to a more aggressive, often fatal, disease phase. Therefore, regulatory mechanisms of stem cell fates have emerged as one of the promising areas for targeted cancer therapies. Recent studies have demonstrated that RNA binding proteins play major roles in both normal and malignant stem cells. We are particularly interested in how RNA binding proteins affect stem cell potentials and contribute to disease development in myeloid leukemia and would like to share our findings on their roles in the branched‐chain amino acid metabolism and stem cell maintenance.Support or Funding InformationSupported by the Research Scholar Grant from the American Cancer Society.

Characterization of Mammary Cells Co‐expressing Separate Lineage Markers

Epithelial cell hierarchy is a highly disputed topic in mammary gland biology. Studying the differentiation potential among cell types within the mammary gland is crucial for understanding normal development and would inform studies in tumorigenesis on a molecular and cellular level. Our recent study uncovers alterations occurring in the aging mouse mammary gland by utilizing single‐cell RNA‐sequencing to study the transcriptomic changes that occur between young and aged mice. From this data, we identified a cluster of luminal cells that expressed canonical lineage markers for two cell types, hormone‐sensing (HS) and alveolar (AV) cells, and that these HS‐AV cells decrease in abundance with age. The co‐expression of these markers contests the presently accepted epithelial hierarchy, as the HS and AV cells are currently thought to be maintained by separate pools of unipotent progenitor cells. Here, we characterized these murine HS‐AV cells by their gene expression pattern consisting of canonical HS and AV markers and confirmed their presence using immunofluorescence staining. In these stainings, we used two sets of canonical HS and AV markers to show co‐localization of both types in single cells, thus uncovering their histological localization within the luminal layer. We further quantified the abundance of these cells and confirmed the population’s decrease with age. Additionally, we have examined human tissues for the presence of HS‐AV cells and have confirmed their existence in the human mammary gland. Future multi‐variate analysis on a larger set of human samples is needed to determine the correlation of HS‐AV cell abundance with age in human mammary glands, as confounding factors such as history of pregnancy, family history, or environmental factors makes it challenging to exclusively analyze the age‐dependent effect on the cell type’s population size. Finally, the lineage relationship of HS‐AV cells to HS and AV cells remains unclear. We are performing functional experiments on isolated HS‐AV cells in order to study their differentiation lineages in vitro as organoid cultures. Unmasking the function of these cells within the normal mammary gland could provide significant insight into the study of the mammary epithelial differentiation hierarchy. A better understanding of this previously less well understood cell type may improve our fundamental knowledge of mammary cell states during normal physiology. Given that the mammary gland is a major organ for cancer development, a more detailed understanding of its cell states may also inform mechanisms of disease development.Support or Funding InformationSusan G. Komen Postdoctoral Fellowship, Croucher Postdoctoral Fellowship (CML), NIH F31 Fellowship (GKG), Hope Funds Postdoctoral Fellowship, Marsha Rivkin Center for Ovarian Cancer Research (YO), Chan Zuckerberg Initiative Donor‐Advised Fund, Silicon Valley Community Foundation, NHGRI Career Development Award (LP), Susan G. Komen Breast Cancer Foundation, and Breast Cancer Research Foundation (JSB).

Differential Alternative Polyadenylation Landscapes Mediate Hematopoietic Stem Cell Activation and Regulate Glutamine Metabolism.

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3' untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies remains elusive. Here, we first demonstrate the requirement of the APA regulator Pabpn1 for hematopoietic stem cell (HSC) function. We then determine the genome-wide APA landscape (APAome) of HSCs and progenitors by performing low-input 3' sequencing paired with bioinformatic pipelines. This reveals transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation. Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching by Nudt21. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal, behavior, and commitment.

Single-Cell Analysis of the Muscle Stem Cell Hierarchy Identifies Heterotypic Communication Signals Involved in Skeletal Muscle Regeneration

Muscle regeneration relies on the regulation of muscle stem cells (MuSCs) through paracrine signaling interactions. We analyzed muscle regeneration in mice using single-cell RNA sequencing (scRNA-seq) and generated over 34,000 single-cell transcriptomes spanning four time-points. We identified 15 distinct cell types including heterogenous populations of muscle stem and progenitor cells. We resolved a hierarchical map of these myogenic cells by trajectory inference and observed stage-specific regulatory programs within this continuum. Through ligand-receptor interaction analysis, we identified over 100 candidate regeneration-associated paracrine communication pairs between MuSCs and non-myogenic cells. We show that myogenic stem/progenitor cells exhibit heterogeneous expression of multiple Syndecan proteins in cycling myogenic cells, suggesting that Syndecans may coordinate myogenic fate regulation. We performed ligand stimulation invitro and confirmed that three paracrine factors (FGF2, TGFβ1, and RSPO3) regulate myogenic cell proliferation in a Syndecan-dependent manner. Our study provides a scRNA-seq reference resource to investigate cell communication interactions in muscle regeneration.

Post-Transcriptional Regulation of Homeostatic, Stressed, and Malignant Stem Cells.

Cellular identity is not driven by differences in genomic content but rather by epigenomic, transcriptomic, and proteomic heterogeneity. Although regulation of the epigenome plays a key role in shaping stem cell hierarchies, differential expression of transcripts only partially explains protein abundance. The epitranscriptome, translational control, and protein degradation have emerged as fundamental regulators of proteome complexity that regulate stem cell identity and function. Here, we discuss how post-transcriptional mechanisms enable stem cell homeostasis and responsiveness to developmental cues and environmental stressors by rapidly shaping the content of their proteome and how these processes are disrupted in pre-malignant and malignant states.