Low-Dose Erythropoietin Amplifies Beneficial Effects of Angiotensin II Blockade on Glomerulosclerosis.

Exogenous erythropoietin (EPO) is used to treat anemia in patients with chronic kidney disease (CKD). Concerns about the possible adverse effect of EPO on the progression of CKD have been raised owing to nonerythroid cell effects. We investigated the effects of low-dose EPO, independent of correcting anemia, on existing glomerulosclerosis. Adult mice underwent 5/6 nephrectomy and were randomized into the following 4 groups at week 8 after surgery: vehicle (VEH), losartan (angiotensin II type 1 receptor blocker [ARB]), darbepoetin-α (DA), or combination (DA+ARB). Four weeks later, mice were euthanized, followed by evaluation of renal structure and function. Glomerular endothelial cells and podocytes were cultured to evaluate the effects of DA on cell migration, apoptosis, and Akt signaling. ARB reduced blood pressure, albuminuria, and the level of serum creatinine and increased hematocrit compared with VEH, whereas low-dose DA only reduced the level of serum creatinine. Combination treatment showed a trend to increase hematocrit and survival compared with ARB alone. Combination treatment but not ARB alone significantly reduced the progression of glomerulosclerosis compared with VEH. Low-dose DA resulted in more preserved glomerular and peritubular capillary endothelial cells with increased p-Akt and even further endothelial cell preservation in combination with ARB. In cultured glomerular endothelial cells, angiotensin II induced more apoptosis, reduced migration, and decreased p-Flk1, a receptor for the proangiogenic vascular endothelial growth factor. DA counteracted these injuries and increased p-Akt, a key factor in angiogenesis and cell survival. DA also protected cultured podocytes against transforming growth factor ß-induced apoptosis and synaptopodin loss. Low-dose EPO directly protects glomerular and peritubular endothelial cells via Akt phosphorylation. Therefore, treatment using a combination of low-dose EPO and ARB results in less progression of glomerulosclerosis in an experimental CKD model.

Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy.

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue.

A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. We previously demonstrated that embryonic stem cells (ESCs) can generate 3D-retina in vitro using a self-organizing stem cell culture technique known as SFEBq. Here we show an optimized culture method for 3D-retina generation from feeder-free human pluripotent stem cells (hPSCs). Although feeder-free hPSC-maintenance culture was suitable for cell therapy, feeder-free hPSC-derived aggregates tended to collapse during 3D-xdifferentiation culture. We found that the initial hPSC state was a key factor and that preconditioning of the hPSC state by modulating TGF-beta and Shh signaling improved self-formation of 3D-neuroepithelium. Using the preconditioning method, several feeder-free hPSC lines robustly differentiated into 3D-retina. In addition, changing preconditioning stimuli in undifferentiated hPSCs altered the proportions of neural retina and retinal pigment epithelium, important quality factors for 3D-retina. We demonstrated that the feeder-free hiPSC-derived 3D-retina differentiated into rod and cone photoreceptors in vitro and in vivo. Thus, preconditioning is a useful culture methodology for cell therapy to direct the initial hPSC state toward self-organizing 3D-neuroepithelium.

Medium- to long-term survival and functional examination of human iPSC-derived retinas in rat and primate models of retinal degeneration.

BACKGROUND: We have previously reported that xeno-transplanted human ESC-derived retinas are able to mature in the immunodeficient retinal degeneration rodent models, similar to allo-transplantations using mouse iPSC-derived retina. The photoreceptors in the latter developed outer segments and formed synapses with host bipolar cells, driving light responses of host retinal ganglion cells. In view of clinical application, here we further confirmed the competency of human iPSC-derived retina (hiPSC-retina) to mature in the degenerated retinas of rat and monkey models. METHODS: Human iPSC-retinas were transplanted in rhodopsin mutant SD-Foxn1 Tg(S334ter)3LavRrrc nude rats and two monkeys with laser-induced photoreceptor degeneration. Graft maturation was studied by immunohistochemistry and its function was examined by multi-electrode array (MEA) recording in rat retinas and visually-guided saccade (VGS) in a monkey. FINDINGS: A substantial amount of mature photoreceptors in hiPSC-retina graft survived well in the host retinas for at least 5 months (rat) to over 2 years (monkey). In 4 of 7 transplanted rat retinas, RGC light responses were detected at the grafted area. A mild recovery of light perception was also suggested by the VGS performance 1.5 years after transplantation in that monkey. INTERPRETATION: Our results support the competency of hiPSC-derived retinas to be clinically applied for transplantation therapy in retinal degeneration, although the light responses observed in the present models were not conclusively distinguishable from residual functions of degenerating host retinas. The functional analysis may be further elaborated using other models with more advanced retinal degeneration.

Effects of combination PPARγ agonist and angiotensin receptor blocker on glomerulosclerosis.

We previously observed that high-dose angiotensin receptor blocker (ARB) can induce regression of existing glomerulosclerosis. We also found that proliferator-activated recepto-γ (PPARγ) agonist can attenuate glomerulosclerosis in a nondiabetic model of kidney disease, with specific protection of podocytes. We now assessed effects of combination therapy with ARB and pioglitazone on established glomerulosclerosis. Sprague-Dawley male rats underwent 5/6 nephrectomy (5/6 Nx) at week 0 and renal biopsy at week 8. Rats were randomized to groups with equal starting moderate glomerulosclerosis, and treated with ARB, PPARγ agonist (pioglitazone), combination or vehicle from weeks 8 to 12. Body weight, systolic blood pressure (SBP), and urinary protein (UP) were measured at intervals. In rats with established sclerosis, SBP, UP, and GS were equal in all groups at week 8 before treatment by study design. Untreated control rats had hypertension, decreased GFR, and progressive proteinuria and glomerulosclerosis at week 12. Only combination therapy significantly ameliorated hypertension and proteinuria. ARB alone or pioglitazone alone had only numerically lower SBP and UP than vehicle at week 12. Both pioglitazone alone and combination had significantly less decline in GFR than vehicle. Combination-induced regression of glomerulosclerosis in more rats from weeks 8 to 12 than ARB or pioglitazone alone. In parallel, combination treatment reduced plasminogen activator inhibitor-1 expression and macrophage infiltration, and preserved podocytes compared with vehicle. These results were linked to increased AT2 receptor and Mas1 mRNA in the combination group. PPARγ agonists in combination with ARB augment regression of glomerulosclerosis, with downregulation of injurious RAAS components vs PPARγ alone, with increased anti-fibrotic/healing RAAS components, enhanced podocyte preservation, and decreased inflammation and profibrotic mechanisms.

Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration.

Retinal transplantation therapy for retinitis pigmentosa is increasingly of interest due to accumulating evidence of transplantation efficacy from animal studies and development of techniques for the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells into retinal tissues or cells. In this study, we aimed to assess the potential clinical utility of hESC-derived retinal tissues (hESC-retina) using newly developed primate models of retinal degeneration to obtain preparatory information regarding the potential clinical utility of these hESC-retinas in transplantation therapy. hESC-retinas were first transplanted subretinally into nude rats with or without retinal degeneration to confirm their competency as a graft to mature to form highly specified outer segment structure and to integrate after transplantation. Two focal selective photoreceptor degeneration models were then developed in monkeys by subretinal injection of cobalt chloride or 577-nm optically pumped semiconductor laser photocoagulation. The utility of the developed models and a practicality of visual acuity test developed for monkeys were evaluated. Finally, feasibility of hESC-retina transplantation was assessed in the developed monkey models under practical surgical procedure and postoperational examinations. Grafted hESC-retina was observed differentiating into a range of retinal cell types, including rod and cone photoreceptors that developed structured outer nuclear layers after transplantation. Further, immunohistochemical analyses suggested the formation of host-graft synaptic connections. The findings of this study demonstrate the clinical feasibility of hESC-retina transplantation and provide the practical tools for the optimization of transplantation strategies for future clinical applications.