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Menstrual-Effluent Exosomes as a Non-Invasive Biopsy for Gynecological Health

 

Group 40: Jessica Wang & Elen Hovhannisyan

Mentor: Dr. Pranav Sharma, Xosomix

 

Project Objective

 

To develop a noninvasive and accurate diagnosis of endometriosis based on in vitro angiogenic and neurogenic activity of menstrual effluent exosomes.

Background

 

• Endometriosis is a chronic condition characterized by lesions of endometrial tissue growing outside the uterus in the pelvic cavity1 affecting >10% of reproductive age .​

• Symptoms include severe pelvic pain, inflammation, lesions, infertility, and buildup of fibrous tissue. ​

• Currently, the only FDA-approved diagnosis is invasive laparoscopy surgery.​

 

Method

 

Volunteers were recruited from the following groups:

• Endometriosis (surgically confirmed)

• Symptomatic control (symptomatic but surgically confirmed healthy)

• Asymptomatic control (healthy)

All volunteers were given a silicone menstrual cup for the collection of menstrual effluent and a 50 mL tube for turning in the sample. The collection was done in the first 1-2 days of menstruation, or when the flow of each participant was heaviest. The samples were submitted to the lab as soon as possible to minimize the chances of contamination or degradation. Once submitted, EDTA was added to prevent clotting, the sample was diluted with DPBS at a 1:2 ratio, and carefully layered over Lymphocyte Separation Medium. After centrifuging the layers, a density gradient was formed, from which plasma and cells were separated and processed individually. The diagram below shows the details of the purification process. Note that exosomes were isolated from plasma.

Flowchart for the purification of exosomes from menstrual effluent.

 

Neurite Outgrowth Assay

 

In order to understand the differences in sensory neuron development from asymptomatic vs endometriosis samples, we must perform a neurite outgrowth assay. We are then able to quantify differences in neurite length between sensory neurons treated with exosomes from different patient cohorts.

 

Human induced pluripotent stem cells (iPSCs) are differentiated into neural crest cells (NCCs) using Stemcell Technologies’ neural crest differentiation kit and the NCCs are then differentiated into mature sensory neurons using Stemcell Technologies’ sensory neuron differentiation and maturation kits. On day 0, 2 million iPSCs were seeded into each well of a 6-well plate and received daily full media changes (2 mL/well) of  STEMdiff™ Neural Crest Differentiation Medium for 6 days. The NCCs are passaged on day 6 and 1 million NCCs are seeded into each well of a 6-well plate. They receive full media change (2 mL/well) every day for 6 days using  STEMdiff™ Sensory Neuron Differentiation Basal Medium and Differentiation Supplement. On day 12, the cells receive a full media change every day (2 mL/well) of BrainPhys™ Neuronal Medium and STEMdiff™ Sensory NeuronMaturation Supplement for 7 days.

 

To validate our differentiation process, we performed immunocytochemistry (ICC) following Stemcell Technologies’ ICC protocol. On day 6, we checked for NCC presence through NGFR and SOX10 marker presence. On day 11, we validated sensory neuron presence using β-III tubulin and peripherin marker presence.

 

 

 

Fluorescence images showing sensory neurons labeled with peripherin (green, right) and β-III tubulin (green, left) markers. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 20µm.​

 

For the neurite growth assay, 50 wells (5 treatment groups of 10 duplicates) of a Corning® 96-well High Content Screening Microplates with Glass Bottom are seeded with 8,125 sensory neurons each. After 2 daily full media changes (2mL/well) of STEMdiff™ Sensory Neuron Differentiation Basal Medium and Differentiation Supplement, the sensory neurons are treated with one of five treatment conditions. 

 

• media (negative control)

• 10 µg/µL BDNF (positive control)

• 20 µg/µL BDNF (positive control)

• 2 µg/well blood-derived exosomes

• 20 µg/well blood-derived exosomes

 

The blood exosomes are used as a proxy for menstrual effluent exosomes. The plates are incubated for 48 hours at 37 ℃. On day four, the sensory neurons are fixed with 4% formaldehyde and stained using lipid dye. Then, the wells are imaged using the BioTek Cytation 5 imaging system. Neurite outgrowth was quantified using Metamorph software by measuring the average neurite length per cell in each treatment group.

 

Flowchart showing neurite outgrowth assay steps

 

Neurite outgrowth quantification

 

Neurite outgrowth was quantified by measuring the average length of the primary neurite per neuron using MetaMorph® software. ​Each neurite was manually traced and logged into an Excel worksheet. The average for each group was calculated from data measured from at least 3 independent wells, with standard error. A p-value was obtained for each BDNF and blood exosome group by comparing to negative control.

 

                    Bipolar neuron with 2 neurites                                Unipolar neuron with 1 neurite

 

 

Neurite Outgrowth Assay Results

 

Varying concentrations of BDNF and blood-derived exosomes are used to treat sensory neurons in a neurite outgrowth assay. After quantitative analysis of average primary neurite lengths from each treatment group, there is a statistically significant increase in neurite length for both high-doses of BDNF (20 µg/µL) and blood-derived exosome (20 µg/well) treatment conditions. Specifically, we found that the 20 µg/µL concentration of blood-derived exosome treatment caused the most increased growth, 659.75 µm, compared to the control length of 468.06 µm.

 

 

 

 

Fluorescence images of neurons (labeled with DiI dye) treated with media (negative control, top), 20 µg/µL BDNF (positive control, middle), or 20 µg/well blood exosomes (bottom). Scale bar = 100µm. ​

 

Angiogenesis Assay

 

Angiogenesis is the formation of new blood vessels, which is known to differ in endometriotic lesions. The goal of this assay is to compare the angiogenic activity signaled by exosomes derived from the menstrual effluent of healthy vs endometriosis patients.

 

Representation of how angiogenesis looks at different levels of activity

 

HUVECs (Human Umbilical Vein Endothelial Cells; ATCC CRL-4053) were cultured over 2-3 days until the correct confluency of cells (50-70%) was achieved. 10ul of Corning® Matrigel® Matrix was plated onto the bottom wells of specialized 96-well plates (ibidi USA, Inc.) as a flat substrate for plating HUVECs. 50 μL of water was added to the outside wells of the 96-well plate to prevent drying of the Matrigel and evaporation of media. The plate with Matrigel was incubated for an hour before cells were added. Before plating, HUVECs were suspended in different concentrations of exosomes, DPBS, and full serum media that served as conditions for the assay. 5 wells of each condition were plated.

• Negative Control - DPBS  (no angiogenic stimulus)

• Positive Control -  Full Serum HUVEC Growth Media

• 0.2 μG exosomes per well

• 2.0 μG exosomes per well

• 5.0 μG exosomes per well

 

The wells were imaged 2 and 4 hours after plating cells. The results were quantified and analyzed using ImageJ and Microsoft Excel. Structures formed by differentiated cells in the wells were categorized into meshes, segments, and branches to represent different levels of angiogenic activity.

 

Representation of how cells form each of the quantified structures

 

 

The structures above were manually traced in every well of cells. The following were calculated for each condition to be compared across different conditions:

• Mesh number

• Mesh Area

• Mesh length

• Segment number

• Segment length

• Average Segment length

• Branch number

• Branch length

• Total segment length (segments + branches)

• Total length (meshes + segments + branches

 

 

References

 

1. Agarwal, Sanjay K., et al. “Clinical Diagnosis of Endometriosis: A Call to Action.” American Journal of Obstetrics and Gynecology, Mosby, 6 Jan. 2019.

 

 

Acknowledgements

 

We’d like to extend a thank you to our mentor, Dr. Pranav Sharma from Xosomix; clinical partner for Xosomix, Dr. Sanjay Agarwal at UC San Diego Health; our TA Meghana Varanasi; and Dr. Alyssa Taylor for their guidance and support throughout this