September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Functional Validation of GLP-grade Induced Pluripotent Stem Cell Derived Retinal Pigment Epithelium: developing a Cell Therapy for AMD.
Author Affiliations & Notes
  • Fnu Ruchi
    NEI, NIH, Bethesda, Maryland, United States
  • Vladimir Khristov
    NEI, NIH, Bethesda, Maryland, United States
  • Balendu Jha
    NEI, NIH, Bethesda, Maryland, United States
  • Dishita Patel
    NEI, NIH, Bethesda, Maryland, United States
  • Qin Wan
    NEI, NIH, Bethesda, Maryland, United States
  • Nathan Hotaling
    NEI, NIH, Bethesda, Maryland, United States
  • Congxiao Zhang
    NEI, NIH, Bethesda, Maryland, United States
  • Kapil Bharti
    NEI, NIH, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   Fnu Ruchi, None; Vladimir Khristov, None; Balendu Jha, None; Dishita Patel, None; Qin Wan, None; Nathan Hotaling, None; Congxiao Zhang, None; Kapil Bharti, None
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 2127. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Fnu Ruchi, Vladimir Khristov, Balendu Jha, Dishita Patel, Qin Wan, Nathan Hotaling, Congxiao Zhang, Kapil Bharti; Functional Validation of GLP-grade Induced Pluripotent Stem Cell Derived Retinal Pigment Epithelium: developing a Cell Therapy for AMD.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2127.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose : AMD, a degenerating eye disease leads to vision loss and blindness in the aging population. Advanced AMD is linked to degeneration of the retinal pigment epithelium (RPE), a pigmented monolayer of epithelial cells which helps in the maintenance of photoreceptors health and functions. Previous work suggests that replacing the damaged RPE with an autologous RPE monolayer can provide potential therapy for AMD. Here, we have describe a GLP complaint induced pluripotent stem (iPS) cell to RPE differentiation protocol that generates functionally mature RPE patch from patient-specifc iPS cells.

Methods : Viral free, foot-print free vectors are used to reprogram patient’s blood cells into GLP-grade iPS cells. iPS cells are karyotyped and exome sequenced to identify potentially tumorigenic mutations and differentiated under xenofree conditions. Flow cytometry and qRT-PCR based assays are used to determine the efficiency of differentiation and purity of cells using RPE markers TYRP1, CRALBP, and BEST1. RPE patch grown on bio-degradable PLGA (poly (lactic-co-glycolic acid) scaffold is validated using electron microscopy (TEM and SEM), immunofluorescence of RPE markers (Ezrin, Collagen IV, RPE65), electrophysiological measurements, and cytokine secretion for VEGF and PEDF.

Results : Karyotypically normal GLP-grade blood cell derived iPS cells were free of any potentially pathological oncogenic mutation. Flow cytometry analysis confirmed that more than 95 percent of differentiating cells were positive for BEST1, CRALBP, and TYRP1. TEM images of RPE patches showed complete polarization with extensive apical processes, apically located melanosomes, and basal infoldings. The electrophysiological measurements of iPS cell-derived RPE monolayer confirm the intactness of the tissue. The trans-epithelial resistance (TER) for monolayer was 400 ohms.cm2 comparable to the primary human RPE. iPS cell derived RPE monolayer secretes cytokines VEGF and PEDF in a polarized fashion with higher VEGF basally and higher PEDF apically.

Conclusions : Research grade developmentally guided differentiation protocol was successfully converted into to GLP compliant manufacturing process. This process sis reproducible and generates pure RPE cells from patient-specific iPS cells. GLP-grade iPSC-RPE monolayers function similar to the native RPE.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×