Characterizing Models to Study Cellular Composition Changes in Response to Pregnancy Hormones in the Human Mammary Gland

Ruiz-Ortiz, Jenelys (May 2023) Characterizing Models to Study Cellular Composition Changes in Response to Pregnancy Hormones in the Human Mammary Gland. PhD thesis, Cold Spring Harbor Laboratory.

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Abstract

The mammary gland is an epithelial structure composed of cells from luminal and basal lineages, all which undergo drastic changes throughout multiple postnatal developmental stages. During female puberty an interplay of Estrogen and Progesterone signaling promote the expansion of the rudimentary mammary epithelial structure into a complex ductal network. The mammary epithelium further matures during pregnancy, where an interplay of Estrogen, Progesterone and Prolactin (EPP) prepares the structure to secrete milk. Parity-associated changes to the mammary gland have been extensively described in mouse models. However, the mammary epithelial structure in mice and humans are both surrounded by different microenvironments, limiting the translational potential of studies done in intact murine mammary glands. Moreover, obtaining human mammary tissue involves invasive surgical procedures, making mammary tissue from women undergoing gestation incredibly difficult to obtain. Therefore, there is a pressing need for scalable systems that we can use to track changes to mammary gland cells in response to controlled signals, in order to advance our understanding of the molecular mechanisms underlying complex developmental stages. Organoids are an emerging culturing system that allow for dynamic tracking of molecular and morphological changes to tissue samples in exposure to controlled developmental signals. Our lab previously demonstrated that EPP treatment can recapitulate the expression of milk associated proteins by organoids derived from murine mammary epithelial cells (MECs), as well as inducing MECs to obtain a parity-associated epigenomic signature. However, further assessment was needed to discern the extent to which hormone treatments reliably recapitulated in vivo developmental stages in organoids. For this work, we used emerging single cell technologies to explore the extent to which MECderived organoids recapitulate in vivo MEC composition, providing an in-depth characterization of this system as a scalable model to study different developmental stages. We first characterized murine MECs without hormone treatment, with different doses of Estrogen to mimic hormone concentration changes during the estrous cycle, and with EPP treatment using single cell RNA-sequencing (scRNA4 seq), and found compositional and transcriptomic changes to MECs associated with hormone response and pregnancy. We further compared the resulting data to previously generated data sets from intact murine mammary tissue collected at different pregnancy stages, and found the acquisition of unique cellular states in vitro. Thus, the results for this portion of the thesis demonstrate the utility and limitations of using organoid systems to dissect the effects of hormones on MEC development. To expand our characterization of models to study mammary gland development, scRNA-seq data sets were generated from mammary tissue samples from healthy women that were never pregnant and that had experienced past pregnancies. Using this approach, it would be possible to obtain a snapshot of persistent changes to the mammary gland occurring after undergoing pregnancy. Preliminary results for this portion of my thesis show MEC compositional differences between tissue from women who had never been pregnant and women who had previous pregnancies, providing a framework for subsequent studies using other human MEC-derived systems. Thus, we then cultured and sequenced organoids from human mammary tissue and treated them with EPP to mimic pregnancy associated development. We showed human MEC-derived organoids are also able to recapitulate compositional and transcriptional changes associated with pregnancy hormones, consequently making organoids a viable system to understand hormone-induced development in human mammary tissue. In order to truly translate our findings on murine MEC-derived organoids and determine conserved mechanisms across evolutionary timescales that contribute to tissue homeostasis during pregnancy, we next set out to compare MEC transcriptional profiles between both murine and human organoids. Similar to previous findings by our lab, we found that, at baseline, progenitor cell types were conserved across species, whereas mature cell types were species-specific. Treatment with EPP resulted in MECs segregating almost exclusively based on species of origin, alluding to MECs becoming highly specialized to species-specific functions in response to pregnancy hormones. Altogether, we have generated a single cell map of murine and human MEC-derived organoids undergoing hormone response and pseudo-pregnancy cycles in vitro. We demonstrate the efficacy of hormone treatments on these specific 3D culture models in recapitulating hormone-driven compositional changes to the mammary epithelial structure. These findings pave the way for future studies to further characterize dynamic changes to specific mammary epithelial sub-populations using these 3D models under different conditions to model primary tissue development.

Item Type:	Thesis (PhD)
Subjects:	bioinformatics bioinformatics > genomics and proteomics Investigative techniques and equipment Investigative techniques and equipment > cell culture > cancer organoids Investigative techniques and equipment > cell culture bioinformatics > genomics and proteomics > small molecules > estrogen diseases & disorders > pregnancy bioinformatics > genomics and proteomics > small molecules
CSHL Authors:	Ruiz-Ortiz, Jenelys
Communities:	CSHL labs > Dos Santos lab School of Biological Sciences > Theses
Depositing User:	Kathleen McGuire
Date:	18 May 2023
Date Deposited:	29 Aug 2024 18:46
Last Modified:	29 Aug 2024 19:08
URI:	https://repository.cshl.edu/id/eprint/41643

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