Kelly E. McKinnon, Spiro Getsios, Teresa K. Woodruff. Distinct Transcriptional Profiles in Engineered Human Ectocervical Tissue Dependent on Menstrual Cycle Phase. (Accepted at Biology of Reproduction).

To investigate genomic pathways that may influence physiology and infectivity during the menstrual cycle, RNA sequence analysis was performed on patient-matched engineered ectocervical tissue after follicular and luteal phase hormone treatments. We developed distinct cellular, molecular and biological profiles in ectocervical epithelium dependent on menstrual cycle phase. Follicular phase hormones were associated with proliferation, transcription, and cell adhesion, while luteal phase samples expressed genes involved in immune cell recruitment, inflammation, and protein modifications. Additionally, our analysis revealed mucins not previously reported in ectocervical tissue, which could play an important role in fertility and disease prevention. This study provides insight into the phenomenon of increased luteal phase vulnerability to infection and identifies potential targets for future research.

 

Kelly E. McKinnon, Rhitwika Sensharma, Chloe Williams, Jovanka Ravix, Spiro Getsios, Teresa K. Woodruff. Development of Ectocervical Tissue Models with Physiologic Endocrine Signaling. (In revision at Biology of Reproduction).

There is a shortage of research models that adequately represent the unique mucosal environment of human ectocervix, limiting development of new therapies for treating infection or cancer. We developed three microphysiologic human ectocervix models to study hormone action during homeostasis. First, we reconstructed ectocervix using decellularized extracellular matrix scaffolds, which supported cell integration and could be clinically useful. Secondly, we generated organotypic systems consisting of ectocervical explants co-cultured with murine ovaries or exogenous hormones, mimicking human menstrual cycles. Additionally, we engineered ectocervix tissue consisting of tissue-specific stromal-equivalents and fully-differentiated epithelium, mimicking in vivo physiology, including squamous maturation, hormone response, and mucin production, which remained viable for 28 days in vitro. The localization of differentiation-dependent mucins in native and engineered tissue was identified for the first time, which will allow increased efficiency in mucin-targeting for drug delivery. In summary, we developed and characterized three microphysiologic human ectocervical tissue models that will be useful for a variety of research applications, including preventative and therapeutic treatments, drug and toxicology studies, and fundamental research on hormone action in a historically understudied tissue that is critical for women’s health.

 

Kelly E. McKinnon**, Emma Gargus**, Hunter Rogers**, Maxwell Edmonds**, Teresa K. Woodruff. Engineering Reproduction. Nature Biomedical Engineering. 2019. (In press).

**denotes equal first author contribution.

In the last few years, a number of remarkable advances have been made in biomedical engineering within the fields of reproductive science and medicine, including engineered reproductive tissues and sophisticated multi-organ in vitro culture systems. These engineered systems are quickly moving the field toward new frontiers that will enable reproductive function beyond current technological limitations. Both male and female reproductive tissues are being engineered as autonomous units and as integrated, multi-organ systems that support germ cell and embryo function, and are capable of phenocopying characteristic endocrine patterns, including the 28-day human ovulatory cycle. The long-term goal of this work is twofold: to develop biomimetic models of human reproductive tissues for research applications and to develop clinical tools that will allow the restoration of reproductive capacity in patients who have lost the parent organ due to disease, iatrogenic effects of medical or surgical treatments, genetic predisposition, or age. With the development of these new technologies, reproductive science is emerging as the next frontier in biomedical engineering, creating new possibilities for not only understanding reproductive biology, but also providing personalized reproductive medicine with transgenerational implications.

 

Kelly E. McKinnon. Microphysiologic Models of Human Ectocervical Tissue to Study Steroid Hormone Action During Homeostasis, Infection, and Oncogenic Transformation. Proquests Dissertation Publishing. 2018.

There is a shortage of research models that adequately represent the unique mucosal environment of human ectocervical tissue, which has limited the development of new therapies for treating infection or cancer. I hypothesized that engineering the microenvironment of ectocervical tissue with in vivo-like endocrine and paracrine support, would enable squamous differentiation and hormone response. An interdisciplinary approach was taken to generate and characterize three distinct microphysiologic tissue culture models to study hormone-action in the human cervix. These microphysiologic models mimicked many aspects of in vivo physiology, including squamous maturation, hormone response, and mucin production, which are important components of barrier defense. Additionally, mRNA transcripts for several mucins not previously reported in the ectocervix and the differentiation-dependent localization of known ectocervical mucins were identified in both native and engineered tissue. To investigate genomic pathways that may influence physiology and infectivity during the menstrual cycle, RNA sequence analysis was performed on patient-matched engineered tissue after follicular and luteal phase hormone treatments. Follicular phase hormones were associated with proliferation, transcription, and cell adhesion, while luteal phase samples expressed genes involved in immune cell recruitment, inflammation, and protein modifications. In summary, I developed three microphysiologic human ectocervical tissue models that differentiate, produce mucins, and respond to hormones, and defined ovarian hormone-action in the human ectocervix during the menstrual cycle, highlighting potential mechanisms that may influence infectivity. This will be useful for a variety of research applications, such as drug development and toxicology studies, development of preventative and therapeutic treatments, and basic research on hormone action in a critical reproductive tissue for women’s health that has been historically understudied.

 

Kelly E. McKinnon**, Shuo Xiao**, Jonathon R. Coppeta**, Jie Zhu**, Hunter Rogers**, Susan A. Olalekan**, Brett C. Isenberg, Danijela Dokic, Alexandra S. Rashedi, Daniel J. Haisenleder, Saurabh S. Malpani, Chanel Arnold-Murray, Kuanwei Chen, Mingyang Jiang, Monica M. Laronda, Thomas Hope, Mary Ellen Pavone, Michael J. Avram, Elizabeth C. Sefton, Spiro Getsios, Joanna Burdette, J. Julie Kim, Jeffrey T. Borenstein, Teresa K. Woodruff. 28-day Menstrual Cycle Hormone Control of Human Reproductive Tract Function in a Microfluidic Culture System. Nature Communications. 2017.

**denotes equal first author contribution.

Altmetric score – 1033 (99^th percentile of articles of similar age in all journals)

National Institute of Environmental and Health Sciences – 2017 Paper of the Year

The endocrine system dynamically controls tissue differentiation and homeostasis, but has not been studied using dynamic tissue culture paradigms. Here we show that a microfluidic system supports murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile, which controls human female reproductive tract and peripheral tissue dynamics in single, dual and multiple unit microfluidic platforms (Solo-MFP, Duet-MFP and Quintet-MPF, respectively). These systems simulate the in vivo female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver, with a sustained circulating flow between all tissues. The reproductive tract tissues and peripheral organs integrated into a microfluidic platform, termed EVATAR, represents a powerful new in vitro tool that allows organ–organ integration of hormonal signalling as a phenocopy of menstrual cycle and pregnancy-like endocrine loops and has great potential to be used in drug discovery and toxicology studies.

 

Monica M. Laronda, Kelly E. McKinnon, Allison Ting, Ann LeFever, Mary B. Zelinski, Teresa K. Woodruff. Good manufacturing practice requirements for the production of tissue vitrification and warming and recovery media for clinical research. Journal of Assisted Reproduction and Genetics. 2017.

Products that are manufactured for use in a clinical trial, with the intent of gaining US Food and Drug Administration (FDA) approval for clinical use, must be produced under an FDA approved investigational new drug (IND) application. We describe work done toward generating reliable methodology and materials for preserving ovarian cortical tissue through a vitrification kit and reviving this tissue through a warming and recovery kit. We have described the critical steps, procedures, and environments for manufacturing products with the intent of submitting an IND. The main objective was to establish an easy-to-use kit that would ensure standardized procedures for quality tissue preservation and recovery across the 117 Oncofertility Consortium sites around the globe. These kits were developed by breaking down the components and steps of a research protocol and recombining them in a way that considers component stability and use in a clinical setting. The kits were manufactured utilizing current good manufacturing practice (cGMP) requirements and environment, along with current good laboratory practices (cGLP) techniques. Components of the kit were tested for sterility and endotoxicity, and morphological endpoint release criteria were established. We worked with the intended down-stream users of these kits for development of the kit instructions. Our intention is to test these initial kits, developed and manufactured here, for submission of an IND and to begin clinical testing for preserving the ovarian tissue that may be used for future restoration of fertility and/or hormone function in women who have gonadal dysgenesis from gonadotoxic treatment regimens or disease.

 

Pope, W. H., Bowman, C.A., Russell, D.A., Jacobs-Sera, D., Asai, D.J, Creswan, S.G., Jacobs, W.R., Hendrix, R.W., Lawrence, J.G., Hatfull, G.F. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife. (2015).

    ** This work included hundreds of undergraduates across the nation in the discovery, sequencing, annotation and report of over 600 phage genomes. My specific contributions were the genome annotations of phages Mufasa and Hope4Ever. Full author list is available as part of the supplementary info.

The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.

 

Yiming Lin, Kelly E. McKinnon, Shin W. Ha, George R. Beck. Inorganic phosphate induces cancer cell mediated angiogenesis dependent on forkhead box protein C2 (FOXC2) regulated osteopontin expression. Molecular Carcinogenesis. (2014). 

Recent studies in both rodents and humans suggest that elevated serum phosphorus, in the context of normal renal function, potentiates, or exacerbates pathologies associates with cardiovascular disease, bone metabolism, and cancer. Our recent microarray studies identified the potent stimulation of pro-angiogenic genes such as forkhead box protein C2 (FOXC2), osteopontin, and Vegfα, among others in response to elevated inorganic phosphate (Pi). Increased angiogenesis and neovascularization are important events in tumor growth and the progression to malignancy and FOXC2 has recently been identified as a potential transcriptional regulator of these processes. In this study we addressed the possibility that a high Pi environment would increase the angiogenic potential of cancer cells through a mechanism requiring FOXC2. Our studies utilized lung and breast cancer cell lines in combination with the human umbilical vascular endothelial cell (HUVEC) vessel formation model to better understand the mechanism(s) by which a high Pi environment might alter cancer progression. Exposure of cancer cells to elevated Pi stimulated expression of FOXC2 and conditioned medium from the Pi-stimulated cancer cells stimulated migration and tube formation in the HUVEC model. Mechanistically, we define the requirement of FOXC2 for Pi-induced osteopontin (OPN) expression and secretion from cancer cells as necessary for the angiogenic response. These studies reveal for the first time that cancer cells grown in a high Pi environment promote migration of endothelial cells and tube formation and in so doing identify a novel potential therapeutic target to reduce tumor progression.