The Influence of Porcine and Axolotl ECM Factors on Human ... · Insoluble Axolotl 5 µg/mL...
Transcript of The Influence of Porcine and Axolotl ECM Factors on Human ... · Insoluble Axolotl 5 µg/mL...
Filtered Soluble
Cells Seededfor 24 Hours
(15,000 cells/well)
Porcine Retinal ECM Factors
Axolotl Retinal ECM Factors
Filtered & Digested Insoluble
Filtered Soluble
Filtered & Digested Insoluble
Protein Estimation
Frozen hRPCs, P6
Cells Cultured in Flasks, P11
Cells Induced with Retinal ECM
for 48 Hours
Plates with Induced Cells
Cell Lysate Collected,Protein Estimation
Bio-Plex Analysis
•Non-Induced Control Cells•Insoluble Porcine•Soluble Porcine•Insoluble Axolotl•Soluble Axolotl•Media Control
The Influence of Porcine and Axolotl ECM Factors on Human Retinal Progenitor CellsAanie Phillips1, Joydip Kundu2, Rebecca Carrier1,2
1Department of Bioengineering, 2Department of Chemical Engineering, Northeastern UniversityData-Driven
Discovery REU
Introduction
Methods
Results
Conclusions
References
Hypothesis: The ECM composition of lower vertebrate retinas can present biochemical cues that trigger cell signaling events which will guide the fate of human retinal progenitor cells (hRPCs) in the retinal regeneration processObjective: Study the effect of retinal ECM factors, isolated from lower vertebrates (axolotl) and higher vertebrates (porcine), on hRPCs
I. Cell Culture and Sample Preparation• ECM factors isolated from porcine and
axolotl retinas• Insoluble ECM factors digested with
pepsin (solubilized)• Protein estimation done to calculate
ECM factor concentrations• Target ECM factor concentrations for
induction: 5 µg/mL and 500 ng/mL
II. Cell Induction• 9 groups, 2 concentrations for each
type of ECM factor
III. Data Analysis• Cells lysed with buffer to release
intracellular proteins• Protein estimation done to calculate
sample concentrations for Bio-Plex• Bio-Plex machine analyzes the
expression of phosphorylated intracellular signaling proteins
Acknowledgements[1] Semba et al., Proteomics 2013, 13, 2500-2511[2] Hamilton et al., Cell Reports 9, 2014, 2056-2070
This study was supported by Northeastern University’s NSF sponsored REU-D3 program, Northeastern University (Tier 1 Provost Grant), and NIH Grant 1R21EY021312 (NEI).
•Many signaling proteins involved with cell growth and differentiation, specifically ERK 1/2, are down-regulated in axolotl ECM induced hRPCs.
•This down-regulation of ERK 1/2 suggests that the cells are maintaining a dedifferentiated state capable of self-renewal2.
•The preservation of self-renewing hRPCs can enable the replenishment of a degenerated retina with appropriate retinal cells.
•Approximately 285 million people worldwide have visual impairments; 39 million are blind. Macular degeneration is a main cause1 (Fig. 1).
•The extracellular matrix (ECM) composition in lower vertebrates is permissive to regeneration in contrast to mammalian matrices in which regeneration does not occur.
•This motivates uncovering the factors essential for successful cell transplantation therapy, a promising treatment approach for retinal degeneration but limited by low survival of implanted cells.
Figure 2. Experiment Overview
Experimental Layout (Fig. 2)
•Expression levels of signaling proteins were assessed with a Bio-Plex MAPK assay (Fig.3).
•ERK 1/2 (responsible for cell differentiation) was down-regulated in axolotl ECM induced hRPCs (Fig. 4).
•A morphological analysis indicated that there were no significant physical differences in the hRPCs across the nine experimental groups.Figure 4. Expression levels of ERK 1/2 across all groups;
a subset of information presented in Fig. 3
ERK 1/2 Expression Levels3500
3000
2500
2000
1500
1000
500
0
Fluo
resc
ence
Inte
nsity
(Abs
olut
e Va
lue) Control Cells
Insoluble Porcine 5 µg/mLInsoluble Porcine 500 ng/mLSoluble Porcine 5 µg/mLSoluble Porcine 500 ng/mLInsoluble Axolotl 5 µg/mLInsoluble Axolotl 500 ng/mLSoluble Axolotl 5 µg/mLSoluble Axolotl 500 ng/mL
Figure 1. Representation of Macular Degeneration (Adapted from Advanced Eye Care)
Normal Macular Degeneration
The Macula is clear and intact in the normal human retina
Drusen, metabolic waste products, cause damage and cell death to retinal cells
Advanced Drug Delivery Research LaboratoryChemical Engineering
ATF-2 ERK 1/2 HSP27 JNK MEK1 p38 MAPK p53 (Ser15) p90RSK Stat3
Figure 3. Expression levels of signaling proteins across all groups
Fluo
resc
ence
Inte
nsity
(Abs
olut
e Va
lue)
4000
3500
3000
2500
2000
1500
1000
500
0
Soluble Porcine 5 µg/mLSoluble Porcine 500 ng/mLInsoluble Axolotl 5 µg/mL
Insoluble Axolotl 500 ng/mLSoluble Axolotl 5 µg/mLSoluble Axolotl 500 ng/mL
Control CellsInsoluble Porcine 5 µg/mLInsoluble Porcine 500 ng/mL