President- Cystinosis Research Foundation

Nancy Stack

Irvine, CA

USA

April5, 2013

Cystinosis Foundation Update 2013

Project title: Differentiation of cystinotic pluripotent stem cells into kidney tissue

We requested one year of seed funding ($46,449) to examine the differentiation of cystinosis-specific human induced pluripotent stem (CTNS-iPS) cells into kidney tissue. CTNS-iPS cells were generated from a nephropathic cystinosis patient as part of a previously funded CRF grant. This work started in Boston, USA at the Center for Regenerative Medicine where Dr Davidson was an Assistant Professor but has been continued at The University of Auckland, New Zealand. CTNS-iPS cells provide new ways to study the cause of cystinosis and to develop cures.

Specific Aim 1: Characterize the formation of renal tissue in cystinotic embryoid bodies by immunofluorescence using a panel of antibodies to renal tubules.

Specific Aim 2: Examine the expression and function of renal progenitor cells from cystinotic embryoid bodies

Background:

Using mouse embryonic stem (ES) cells we identified a specific set of culture conditions whereby ES cells can be induced to express renal progenitor markers. In this protocol, we first treat mouse ES cells with BMP4 and the Wnt antagonist secreted frizzled related protein-2 (SFRP2) then purify Flk1+ mesodermal cells using magnetic beads. After a further 3 days of culture in the presence of BMP4 and SRFP2 the Flk1-derived cells were found to express the renal progenitor markers Odd1, WT1, Six2, Eya1, and Sall1. In the current proposal, one of our goals was to translate this protocol to human CTNS-iPS cells so that ultimately, we can generate cystinotic proximal tubule cells.

Progress to date:

To date, we have focused predominately on Specific Aim 2. Our initial attempts to reproduce the protocol that works with mouse ES cells were not successful with CTNS-iPS cells and we obtained variable results when renal progenitor markers (Six2, Osr1, Lhx1, Pax2) were examined by qPCR. During the course of this work, two papers were published that report the formation of renal progenitors and proximal tubule-like cells from human pluripotent stem cells (Mae et al., Nature Comm. 2012; Narayanan et al. Kidney Int. 2012). Based on their success, we have modified our approach and adopted the addition of the BMP7 and Activin growth factors into our protocol. We are also reproducing the Narayanan et al. method to determine whether we can mature CTNS-iPS cells directly to a proximal tubule cell state. If so, we will focus our efforts on characterizing CTNS-iPS-derived proximal tubules (presence of enlarged lysosomes, cystine loading, sensitivity to apoptosis etc) as the generation of these cells is our ultimate goal.

Sincerely,

Alan J. Davidson