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