Proliferative Study of Adipose-Derived Stem

Cells

Jay SehgalNorth Allegheny Senior

High SchoolGrade 11

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

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

Introduction

ASCs derived from: Abdominal superficial fat

Subcutaneous Above abdominal muscles

Omental fat Omentum Deep in abdomen

Which depot is better for clinical applications?

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

Hypothesis

Null hypothesis Stem cells derived from omental

fat will have an equal proliferative capacity as stem cells derived from abdominal superficial fat

Key Materials

CyQUANT® Cell Proliferation Assay Kit

Hemacytometer Human abdominal superficial fat Human omental fat SpectraFluor fluorescence reader

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

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

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

67F Omental – 24hrs

100x

67F Abd. Sup. – 24hrs

100x

67F Omental – 48hrs

100x

67F Abd. Sup. – 48hrs

100x

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

DataAverage Cell Counts

2,500 cells Omental Sup. Abd. p-value

24 hours3810.78 1745.75 0.095

48 hours4357.09 4685.33 0.837

96 hours9937.68 11155.51 0.709

10,000 cells Omental Sup. Abd. p-value

24 hours7895.20 6520.62 0.423

48 hours10770.66 9981.24 0.662

96 hours29716.99 45901.66 0.251

40,000 cells Omental Sup. Abd. p-value

24 hours18669.17 9168.58 0.098

48 hours15231.59 15316.03 0.938

96 hours31352.90 35671.46 0.736

DataAverage Cell Counts

20 30 40 50 60 70 80 90 1000

2000

4000

6000

8000

10000

12000

2,500 cells

OmentalSup. Abd.

time (hrs)

# of

cel

ls

DataAverage Cell Counts

20 30 40 50 60 70 80 90 1000

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

10,000 cells

OmentalSup. Abd.

time (hrs)

# of

cel

ls

DataAverage Cell Counts

20 30 40 50 60 70 80 90 1000

5000

10000

15000

20000

25000

30000

35000

40000

40,000 cells

OmentalSup. Abd.

time (hrs)

# of

cel

ls

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

Possible Sources of Error

Sample size Standard curve Initial count w/ hemacytometer Calibration of equipment

SpectraFluor Pipettes

Future Research

Further superficial abdominal vs. omental assays include: Differentiation

Adipogenesis (fat) Chondrogenesis (cartilage)

Apoptosis (PCD)