Clonal tracking in cancer and metastasis.

The eradication of many cancers has proven challenging due to the presence of functionally and genetically heterogeneous clones maintained by rare cancer stem cells (CSCs), which contribute to disease progression, treatment refractoriness, and late relapse. The characterization of functional CSC activity has necessitated the development of modern clonal tracking strategies. This review describes viral-based and CRISPR-Cas9-based cellular barcoding, lineage tracing, and imaging-based approaches. DNA-based cellular barcoding technology is emerging as a powerful and robust strategy that has been widely applied to in vitro and in vivo model systems, including patient-derived xenograft models. This review also highlights the potential of these methods for use in the clinical and drug discovery contexts and discusses the important insights gained from such approaches.

The Mayo Clinic Salivary Tissue-Organoid Biobanking: A Resource for Salivary Regeneration Research.

The salivary gland (SG) is an essential organ that secretes saliva, which supports versatile oral function throughout life, and is maintained by elusive epithelial stem and progenitor cells (SGSPC). Unfortunately, aging, drugs, autoimmune disorders, and cancer treatments can lead to salivary dysfunction and associated health consequences. Despite many ongoing therapeutic efforts to mediate those conditions, investigating human SGSPC is challenging due to lack of standardized tissue collection, limited tissue access, and inadequate purification methods. Herein, we established a diverse and clinically annotated salivary regenerative biobanking at the Mayo Clinic, optimizing viable salivary cell isolation and clonal assays in both 2D and 3D-matrigel growth environments. Our analysis identified ductal epithelial cells in vitro enriched with SGSPC expressing the CD24/EpCAM/CD49f+ and PSMA- phenotype. We identified PSMA expression as a reliable SGSPC differentiation marker. Moreover, we identified progenitor cell types with shared phenotypes exhibiting three distinct clonal patterns of salivary differentiation in a 2D environment. Leveraging innovative label-free unbiased LC-MS/MS-based single-cell proteomics, we identified 819 proteins across 71 single cell proteome datasets from purified progenitor-enriched parotid gland (PG) and sub-mandibular gland (SMG) cultures. We identified distinctive co-expression of proteins, such as KRT1/5/13/14/15/17/23/76 and 79, exclusively observed in rare, scattered salivary ductal basal cells, indicating the potential de novo source of SGSPC. We also identified an entire class of peroxiredoxin peroxidases, enriched in PG than SMG, and attendant H2O2-dependent cell proliferation in vitro suggesting a potential role for PRDX-dependent floodgate oxidative signaling in salivary homeostasis. The distinctive clinical resources and research insights presented here offer a foundation for exploring personalized regenerative medicine.

Puberty Blocker and Aging Impact on Testicular Cell States and Function.

Spermatogonial stem cell (SSC) acquisition of meiotogenetic state during puberty to produce genetically diverse gametes is blocked by drugs collectively referred as 'puberty blocker' (PB). Investigating the impact of PB on juvenile SSC state and function is challenging due to limited tissue access and clinical data. Herein, we report largest clinically annotated juvenile testicular biorepository with all children with gender dysphoria on chronic PB treatment highlighting shift in pediatric patient demography in US. At the tissue level, we report mild-to-severe sex gland atrophy in PB treated children. We developed most extensive integrated single-cell RNA dataset to date (>100K single cells; 25 patients), merging both public and novel (52 month PB-treated) datasets, alongside innovative computational approach tailed for germ cells and evaluated the impact of PB and aging on SSC. We report novel constitutional ranges for each testicular cell type across the entire age spectrum, distinct effects of treatments on prepubertal vs adult SSC, presence of spermatogenic epithelial cells exhibiting post-meiotic-state, irrespective of age, puberty status, or PB treatment. Further, we defined distinct effects of PB and aging on testicular cell lineage composition, and SSC meiotogenetic state and function. Using single cell data from prepubertal and young adult, we were able to accurately predict sexual maturity based both on overall cell type proportions, as well as on gene expression patterns within each major cell type. Applying these models to a PB-treated patient that they appeared pre-pubertal across the entire tissue. This combined with the noted gland atrophy and abnormalities from the histology data raise a potential concern regarding the complete 'reversibility' and reproductive fitness of SSC. The biorepository, data, and research approach presented in this study provide unique opportunity to explore the impact of PB on testicular reproductive health.

Pathogenic BRCA1 variants disrupt PLK1-regulation of mitotic spindle orientation.

Preneoplastic mammary tissues from human female BRCA1 mutation carriers, or Brca1-mutant mice, display unexplained abnormalities in luminal differentiation. We now study the division characteristics of human mammary cells purified from female BRCA1 mutation carriers or non-carrier donors. We show primary BRCA1 mutant/+ cells exhibit defective BRCA1 localization, high radiosensitivity and an accelerated entry into cell division, but fail to orient their cell division axis. We also analyse 15 genetically-edited BRCA1 mutant/+ human mammary cell-lines and find that cells carrying pathogenic BRCA1 mutations acquire an analogous defect in their division axis accompanied by deficient expression of features of mature luminal cells. Importantly, these alterations are independent of accumulated DNA damage, and specifically dependent on elevated PLK1 activity induced by reduced BRCA1 function. This essential PLK1-mediated role of BRCA1 in controlling the cell division axis provides insight into the phenotypes expressed during BRCA1 tumorigenesis.

Genesis of fecal floatation is causally linked to gut microbial colonization in mice.

The origin of fecal floatation phenomenon remains poorly understood. Following our serendipitous discovery of differences in buoyancy of feces from germ-free and conventional mice, we characterized microbial and physical properties of feces from germ-free and gut-colonized (conventional and conventionalized) mice. The gut-colonization associated differences were assessed in feces using DNA, bacterial-PCR, scanning electron microscopy, FACS, thermogravimetry and pycnometry. Based on the differences in buoyancy of feces, we developed levô in fimo test (LIFT) to distinguish sinking feces (sinkers) of germ-free mice from floating feces (floaters) of gut-colonized mice. By simultaneous tracking of microbiota densities and gut colonization kinetics in fecal transplanted mice, we provide first direct evidence of causal relationship between gut microbial colonization and fecal floatation. Rare discordance in LIFT and microbiota density indicated that enrichment of gasogenic gut colonizers may be necessary for fecal floatation. Finally, fecal metagenomics analysis of 'floaters' from conventional and syngeneic fecal transplanted mice identified colonization of > 10 gasogenic bacterial species including highly prevalent B. ovatus, an anaerobic commensal bacteria linked with flatulence and intestinal bowel diseases. The findings reported here will improve our understanding of food microbial biotransformation and gut microbial regulators of fecal floatation in human health and disease.

DNA barcoded competitive clone-initiating cell analysis reveals novel features of metastatic growth in a cancer xenograft model.

A problematic feature of many human cancers is a lack of understanding of mechanisms controlling organ-specific patterns of metastasis, despite recent progress in identifying many mutations and transcriptional programs shown to confer this potential. To address this gap, we developed a methodology that enables different aspects of the metastatic process to be comprehensively characterized at a clonal resolution. Our approach exploits the application of a computational pipeline to analyze and visualize clonal data obtained from transplant experiments in which a cellular DNA barcoding strategy is used to distinguish the separate clonal contributions of two or more competing cell populations. To illustrate the power of this methodology, we demonstrate its ability to discriminate the metastatic behavior in immunodeficient mice of a well-established human metastatic cancer cell line and its co-transplanted LRRC15 knockdown derivative. We also show how the use of machine learning to quantify clone-initiating cell (CIC) numbers and their subsequent metastatic progeny generated in different sites can reveal previously unknown relationships between different cellular genotypes and their initial sites of implantation with their subsequent respective dissemination patterns. These findings underscore the potential of such combined genomic and computational methodologies to identify new clonally-relevant drivers of site-specific patterns of metastasis.

Assays for functionally defined normal and malignant mammary stem cells.

The discovery of rare, heterogeneous self-renewing stem cells with shared developmental and molecular features within epithelial components of mammary gland and breast cancers has provided a conceptual framework to understand cellular composition of these tissues and mechanisms that control their number. These normal mammary epithelial stem cells (MaSCs) and breast cancer stem cells (BCSCs) were identified and analyzed using transplant assays (namely mammary repopulating unit (MRU) assay, mammary tumor-initiating cell (TIC) assay), which reveal their latent ability to regenerate respective normal and malignant epithelial tissues with self-renewing units displaying hierarchical cellular differentiation over multiple generations in recipient mice. "Next-generation" methods using "barcoded" normal and malignant mammary cells, with the help of next-generation sequencing (NGS) technology, have revealed hidden complexity and heterogeneous growth potential of MaSCs and BCSCs. Several single markers or combinations of markers have been reported to prospectively enrich MaSCs and BCSCs. Such markers and the extent to which they enrich for MaSCs and BCSCs activity require a critical appraisal. Also, knowledge of the functional assays and their limitations and harmonious reporting of results is a prerequisite to improve our understanding of MaSCs and BCSCs. This chapter describes evolution of the concept of MaSCs and BCSCs, and specific methodologies to investigate them.

Systematic evaluation of markers used for the identification of human induced pluripotent stem cells.

Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4+ and TRA-1-60+ cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs).

Understanding the Molecular Basis of Heterogeneity in Induced Pluripotent Stem Cells.

Reprogramming of somatic cells to generate induced pluripotent stem cells (iPSCs) has considerable latency and generates epigenetically distinct partially and fully reprogrammed clones. To understand the molecular basis of reprogramming and to distinguish the partially reprogrammed iPSC clones (pre-iPSCs), we analyzed several of these clones for their molecular signatures. Using a combination of markers that are expressed at different stages of reprogramming, we found that the partially reprogrammed stable clones have significant morphological and molecular heterogeneity in their response to transition to the fully pluripotent state. The pre-iPSCs had significant levels of OCT4 expression but exhibited variable levels of mesenchymal-to-epithelial transition. These novel molecular signatures that we identified would help in using these cells to understand the molecular mechanisms in the late of stages of reprogramming. Although morphologically similar mouse iPSC clones showed significant heterogeneity, the human iPSC clones isolated initially on the basis of morphology were highly homogeneous with respect to the levels of pluripotency.