1. Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11

2. Background Information  Stem cells  Undifferentiated cells  Pluripotent  Mature into specialized cells  Embryonic vs. Adult  Embryonic  Controversial (destruction of embryo)  Adult  Non-controversial  Self-renewing  Great therapeutic potential

3. Background Information  Adipose-derived stem cells (ASCs)  Isolated from human adipose tissue (fat)  Abundant - Ideal source for regenerative medicine use  Plastic surgery  Liposuction  Human ASCs  Bone  Cartilage  Muscle  Fat

4. Introduction  ASCs derived from:  Abdominal superficial fat  Subcutaneous  Above abdominal muscles  Omental fat  Omentum  Deep in abdomen  Which depot is better for clinical applications?

5. Purpose  Anecdotal evidence suggests omental fat stem cells have a greater proliferative capacity than those derived from other depots  Goal of this study is to confirm or deny this theory

6. Hypothesis  Null hypothesis  Stem cells derived from omental fat will have an equal proliferative capacity as stem cells derived from abdominal superficial fat

7. Key Materials  CyQUANT® Cell Proliferation Assay Kit  Hemacytometer  Human abdominal superficial fat  Human omental fat  SpectraFluor fluorescence reader

8. Isolation & Expansion  Fat tissue from plastic surgeon  2 patients  67F & 52F  Abdominal & Omental depots  ASCs isolated from fat into ASC growth media  ASCs cultured  Incubator @ 37°C  1-2 weeks

9. Initial Count  ASCs:  Rinsed in phosphate buffered saline (PBS) w/ ethylenediaminetetraacetic acid (EDTA) to disengage clumped cells  Suspended w/ trypsin  Centrifuged to form pellet  Supernatant (w/ trypsin) removed  Media added to pellet  ASCs vortexed to re-suspend  Stained w/ Trypan blue  Counted using hemacytometer  Pellet w/ set # of cells frozen for each depot

10. Proliferation  ASCs for each patient & depot incubated at 3 time points at 3 seating densities in triplicate  24, 48, 96 hrs  2.5k, 10k, 40k cells  At each time point  Media aspirated  ASCs rinsed in PBS  Plate frozen to lyse cell membranes  DNA preserved

11. 67F Omental – 24hrs 100x

12. 67F Abd. Sup. – 24hrs 100x

13. 67F Omental – 48hrs 100x

14. 67F Abd. Sup. – 48hrs 100x

15. CyQUANT® Assay  Frozen pellet used to create standard curve  Cells added from suspended pellet to 16 well plate w/ increasing cell #s  Cells thawed & lysed w/ buffer  Green fluorescent dye, CyQUANT® GR dye, added to cells  Exhibits strong fluorescence enhancement when bound to cellular nucleic acids  Std. curve plates read in SpectraFluor fluorescence reader  CyQUANT® GR dye & cell-lysis buffer added to patient cells  Fluorescence read in SpectraFluor  Readings compared to std. curve to get cell count

16. Data Average Cell Counts 2,500 cellsOmentalSup. Abd.p-value 24 hours 3810.781745.750.095 48 hours 4357.094685.330.837 96 hours 9937.6811155.510.709 10,000 cellsOmentalSup. Abd.p-value 24 hours 7895.206520.620.423 48 hours 10770.669981.240.662 96 hours 29716.9945901.660.251 40,000 cellsOmentalSup. Abd.p-value 24 hours 18669.179168.580.098 48 hours 15231.5915316.030.938 96 hours 31352.9035671.460.736

17. Data Average Cell Counts

20. Data Analysis/Conclusion  Trends show abdominal superficial cells proliferate faster  P-values of the t-tests state data is statistically insignificant  p-value > 0.05  Insufficient evidence to reject the null hypothesis

21. Possible Sources of Error  Sample size  Standard curve  Initial count w/ hemacytometer  Calibration of equipment  SpectraFluor  Pipettes

22. Future Research  Further superficial abdominal vs. omental assays include:  Differentiation  Adipogenesis (fat)  Chondrogenesis (cartilage)  Apoptosis (PCD)