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Safer and Better? Alternative Reprogramming Technique Used for Glaucoma Cell Replacement Therapy

Review of “Continuous non-cell autonomous reprogramming to generate retinal ganglion cells for glaucomatous neuropathy” from Stem Cells by Stuart P. Atkinson

Current therapeutic strategies for glaucoma, the most common cause of irreversible blindness [1], act only to manage the condition and there are currently no means to replace the retinal ganglion cells (RGCs) which are lost in this disease. While pluripotent stem cell-based therapies are an exciting option, this approach has several barriers to its application, although Iqbal Ahmad (University of Nebraska Medical Center, USA) has now proposed the use of a “non‐cell autonomous” reprogramming strategy as a new and improved therapeutic approach [2]. This reprogramming methodology dispenses with the standard nucleic acid-mediated strategy and instead treats cells with conditioned media (CM) designed to mimic the primitive embryonic environment. This is posited to create a safer and more robust source of induced pluripotent stem cells (iPSCs) from patient samples for subsequent differentiation towards transplantable RGCs. Now, in a study published in Stem Cells, the Ahmad group report on the creation of RGCs from adult corneal limbal cells using this methodology, and demonstrate iPSC-derived RGCs to be functional and safe in transplantation experiments [3].

Initial reprogramming efforts used adult mouse limbal progenitors (epithelial cells which express Sox2, Klf4, and Myc) which the group reprogrammed to iPSCs using embryonic stem cell CM (ESCM) supplemented with MAPK and GSK3 inhibitors as previously described [4, 5]. Pluripotent colonies first emerged after 20 days, and to derive retinal progenitor cells (RPCs), the researchers then differentiated iPSCs as embryoid bodies (EBs) in neuronal induction medium containing Noggin to produce cells which expressed neuroectodermal progenitor markers (Nestin and Musashi) and the pro‐neural transcription factor Mash1. A further 25 days in medium containing FGF2 and Noggin mediated the derivation and expansion of RPCs which had a similar global gene expression pattern to cells of the embryonic day 14 (E14) mouse retina.

The generation of RGCs from RPCs utilized CM obtained from embryonic day 14 rat retinal cells, a time point which represents the peak of RGC generation in the rodent retina. After 15 days, a subset of cells expressed regulators of RGC differentiation (Atoh7, Brn3b, and Islet1), while a smaller subset co-expressed Brn3b and the mature RGC marker Thy1, thus confirming a RGC identity. Functional assessment of Brn3b/Thy1 cells using whole cell patch recordings found similar electrophysiological characteristics between iPSC-derived RGCs and those isolated from rat and primate retina. iPSC-derived RGCs also expressed genes involved in guiding RGC axons to their specific targets, and when cultured on aggregates made from mouse midbrain, RGCs elaborated long processes that were oriented towards superior colliculus (SC) cells, the target of RGC axons in the midbrain, but not inferior colliculus (IC) cells, a target of the auditory neurons.

Finally, the researchers assessed the ability of RGCs to integrate within the retina using Morrison’s rat model of ocular hypertension [6]. Two weeks after intravitreal transplant, a small subset of Brn3b-postivie cells had integrated into the host retina while apical processes from these transplanted RGCs directed themselves towards the inner nuclear layer where RGCs receive input from bipolar cells. Additionally, iPSC-derived RGCs injected subcutaneously into immune‐deficient mice did not form teratomas, suggesting that they are safe in the long-term.

Overall, this simple technique (see figure for overview) produces therapeutically relevant cells in a safe and efficacious manner, which may be used in in vitro models for the investigation of glaucoma disease mechanisms, RGC development, drug testing, and may in the future lead to a treatment of glaucomatous neuropathy. The epithelial nature of limbal cells, as well as the expression of 3 of the 4 (just lacking Oct4!) classic reprogramming factors makes limbal progenitors an enticing target for ocular therapies. Further studies may address whether differentiation of these limbal-iPSCs to a non-related cell type provides suitably functional cells, and therefore, if they represent an ideal source for therapeutic purposes throughout the body. Alternatively, the identification of other similar progenitors from distinct tissues may aid reprogramming efforts and bring about safer, cheaper, and better therapeutic options.

Discussion Points
• Do transplanted cells mediate any type of functional recovery?
• Can ESC-derived CM, and CM used for differentiation purposes, be replaced by an animal free defined medium?
• Is this strategy amenable to industrial level scale up to provide sufficient cell numbers for therapeutic use?
• What other somatic cell types are amenable to “non‐cell autonomous” reprogramming?
• Do iPSCs created in this manner still retain an “epigenetic memory” which may affect differentiation propensities?

1. Coleman AL Glaucoma. Lancet 1999;354:1803-1810.
2. Parameswaran S, Balasubramanian S, Rao MS, et al. Concise review: non-cell autonomous reprogramming: a nucleic acid-free approach to induction of pluripotency. Stem Cells 2011;29:1013-1020.
3. Parameswaran S, Dravid SM, Teotia P, et al. Continuous non-cell autonomous reprogramming to generate retinal ganglion cells for glaucomatous neuropathy. Stem Cells 2015;33:1743-1758.
4. Balasubramanian S, Babai N, Chaudhuri A, et al. Non cell-autonomous reprogramming of adult ocular progenitors: generation of pluripotent stem cells without exogenous transcription factors. Stem Cells 2009;27:3053-3062.
5. Parameswaran S, Balasubramanian S, Babai N, et al. Nucleic acid and non-nucleic acid-based reprogramming of adult limbal progenitors to pluripotency. PLoS One 2012;7:e46734.
6. Morrison JC, Moore CG, Deppmeier LM, et al. A rat model of chronic pressure-induced optic nerve damage. Experimental eye research 1997;64:85-96.