Understanding Dynamic Behavior of Embryonic Stem Cells

Shubham DebnathUniversity of Minnesota-Twin Cities

debna002@umn.edu

Advisor: Dr. Bir BhanuBRITE REU 2009

University of California-Riverside

Overview

• Introduction to embryonic stem cells and importance• Description of video processing and image segmentation

methods used for study• Stem cell videos used for data and examples of use of

segmentation and image analysis methods• Results and Analysis• Conclusions• Future pursuits

Stem Cells and Importance

• Derived from the inner cell mass of early stage embryos, known as blastocysts

• Known to be pluripotent and can differentiate into a variety of cell types

• Very important towards study in the future of medicine and healthcare

Stem Cells and Importance• Attach to substrate to

differentiate based on the environment they are placed in

• For mitosis to proceed, cells must unattach themselves, divide, then reattach.

• Behavior of embryonic stem cells is not fully understood

• Past and continued research at Stem Cell Center at UCR• Effects of smoke and alcohol

on stem cell behavior

Mitosis• Process by which eukaryotic

cells divide into two identical daughter cells

• Consists of various phases in which the nucleus and cytoplasm divide ending with cytokinesis and cleavage into two cells

• Importance for maintenance of genome set

• Rate of mitosis depends on tissue renewal for stem cells; varies for different cell types

Introduction to Methods• Otsu’s Algorithm for Binary

Thresholding:

• Inputs a grey-scale image, automatically finds a threshold value, splits the image accordingly

• Threshold value is found by histogram analysis

• Outputs a binary image showing regions of interest

• Connected Components Analysis:

• Only done on binary images• For each pixel, checks

neighboring pixels and labels each region accordingly

• Labeled with random pseudo colors for visual identification of each connected component

Otsu’s Algorithm

Threshold Value: 129

Otsu’s Algorithm

Original Image Segmented Image

Connected Components

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Binary Image Result of Connected Components Algorithm

Examples

Video of Stem Cells

Hypotheses• Number of mitosis occurrences: approximately 6-10• Cell count should be similar and beginning and end of video

– Cells going through mitosis, attaching, apoptotic events– If recorded over a longer period of time, cell count remains

essentially the same• Large jumps, spikes in data represent colonies of cells unattaching

together, multiplying• Sharp drop shows colony reattaching to substrate• Low number of unattached cells should correspond to low number of

pixels in “white” membranes– More pixels in darker mass of cell showing how surface area

increases with attachment to substrate

Plate 9 – 20X

Results

Results

Plate 6 – 20X

Results

Results

Analysis

• Graphs complement each other – directly related

• Spikes in graphs accurately show points of mitosis

• Shows how surface area of cell changes with attachment and cell division

• Problems with counting come with colonies of cells

• Background noise, light

Conclusions

• Video processing can be used for the segmentation of stem cell videos for their characterization.

• Mitosis is important cell differentiation in stem cells and for regulation of processes in the human body

• Mitosis count between 6 and 10 divisions based on resulting graphs– Watching videos agrees with these estimations

• Time: 4 to 6 frames for process of mitosis to start and complete– 8 to 12 minutes

• Colonies tend to unattach and multiply together

Future Research

• Use of the relaxation gradient algorithm• Choosing of different thresholds• Use of a new Nikon Biostation can be used to record videos for

longer times and with higher magnification and resolution.• More biomedical engineering based objective: behavior can be

simulated computationally with macromolecular interactions• The free energy of cells in various states can be calculated to

find a minimum at which the cell responds to diverse changes.

Acknowledgements• Special thanks to my research

advisor, Professor Bir Bhanu of UC-Riverside

• Students at Center for Research in Intelligent System (CRIS)

• Thanks to Dr. Prue Talbot and students at the Stem Cell Center at UCR for providing the data

• Thanks to the BRITE REU program funded by the National Science Foundation (NSF)

Questions?