Sarah J. Dickerson

Blood-Derived Human iPS Cells Generate Optic Vesicle–Like Structures with the Capacity to Form Retinal Laminae and Develop Synapses

PURPOSE We sought to determine if human induced pluripotent stem cells (iPSCs) derived from blood could produce optic vesicle-like structures (OVs) with the capacity to stratify and express markers of intercellular communication. METHODS Activated T-lymphocytes from a routine peripheral blood sample were reprogrammed by retroviral transduction to iPSCs. The T-lymphocyte-derived iPSCs (TiPSCs) were characterized for pluripotency and differentiated to OVs using our previously published protocol. TiPSC-OVs were then manually isolated, pooled, and cultured en masse to more mature stages of retinogenesis. Throughout this stepwise differentiation process, changes in anterior neural, retinal, and synaptic marker expression were monitored by PCR, immunocytochemistry, and/or flow cytometry. RESULTS TiPSCs generated abundant OVs, which contained a near homogeneous population of proliferating neuroretinal progenitor cells (NRPCs). These NRPCs differentiated into multiple neuroretinal cell types, similar to OV cultures from human embryonic stem cells and fibroblast-derived iPSCs. In addition, portions of some TiPSC-OVs maintained their distinctive neuroepithelial appearance and spontaneously formed primitive laminae, reminiscent of the developing retina. Retinal progeny from TiPSC-OV cultures expressed numerous genes and proteins critical for synaptogenesis and gap junction formation, concomitant with the emergence of glia and the upregulation of thrombospondins in culture. CONCLUSIONS We demonstrate for the first time that human blood-derived iPSCs can generate retinal cell types, providing a highly convenient donor cell source for iPSC-based retinal studies. We also show that cultured TiPSC-OVs have the capacity to self-assemble into rudimentary neuroretinal structures and express markers indicative of chemical and electrical synapses.

Human lymphoblastoid B cell lines reprogrammed to EBV-free induced pluripotent stem cells

Generation of patient-specific induced pluripotent cells (iPSCs) holds great promise for regenerative medicine. Epstein-Barr virus immortalized lymphoblastoid B-cell lines (LCLs) can be generated from a minimal amount of blood and are banked worldwide as cellular reference material for immunologic or genetic analysis of pedigreed study populations. We report the generation of iPSCs from 2 LCLs (LCL-iPSCs) via a feeder-free episomal method using a cocktail of transcription factors and small molecules. LCL-derived iPSCs exhibited normal karyotype, expressed pluripotency markers, lost oriP/EBNA-1 episomal vectors, generated teratomas, retained donor identity, and differentiated in vitro into hematopoietic, cardiac, neural, and hepatocyte-like lineages. Significantly, although the parental LCLs express viral EBNA-1 and other Epstein-Barr virus latency-related elements for their survival, their presence was not detectable in LCL-iPSCs. Thus, reprogramming LCLs could offer an unlimited source for patient-specific iPSCs.

Modeling Human Retinal Development with Patient‐Specific Induced Pluripotent Stem Cells Reveals Multiple Roles for Visual System Homeobox 2

Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate along the retinal lineage in a manner that mimics normal mammalian development. Under certain culture conditions, hiPSCs form optic vesicle‐like structures (OVs), which contain proliferating progenitors capable of yielding all neural retina (NR) cell types over time. Such observations imply conserved roles for regulators of retinogenesis in hiPSC‐derived cultures and the developing embryo. However, whether and to what extent this assumption holds true has remained largely uninvestigated. We examined the role of a key NR transcription factor, visual system homeobox 2 (VSX2), using hiPSCs derived from a patient with microphthalmia caused by an R200Q mutation in the VSX2 homeodomain region. No differences were noted between (R200Q)VSX2 and sibling control hiPSCs prior to OV generation. Thereafter, (R200Q)VSX2 hiPSC‐OVs displayed a significant growth deficit compared to control hiPSC‐OVs, as well as increased production of retinal pigmented epithelium at the expense of NR cell derivatives. Furthermore, (R200Q)VSX2 hiPSC‐OVs failed to produce bipolar cells, a distinctive feature previously observed in Vsx2 mutant mice. (R200Q)VSX2 hiPSC‐OVs also demonstrated delayed photoreceptor maturation, which could be overcome via exogenous expression of wild‐type VSX2 at early stages of retinal differentiation. Finally, RNAseq analysis on isolated hiPSC‐OVs implicated key transcription factors and extracellular signaling pathways as potential downstream effectors of VSX2‐mediated gene regulation. Our results establish hiPSC‐OVs as versatile model systems to study retinal development at stages not previously accessible in humans and support the bona fide nature of hiPSC‐OV‐derived retinal progeny. Stem Cells 2014;32:1480–1492

Modeling human retinal development with patient-specific iPS cells reveals multiple roles for VSX2

Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate along the retinal lineage in a manner that mimics normal mammalian development. Under certain culture conditions, hiPSCs form optic vesicle‐like structures (OVs), which contain proliferating progenitors capable of yielding all neural retina (NR) cell types over time. Such observations imply conserved roles for regulators of retinogenesis in hiPSC‐derived cultures and the developing embryo. However, whether and to what extent this assumption holds true has remained largely uninvestigated. We examined the role of a key NR transcription factor, visual system homeobox 2 (VSX2), using hiPSCs derived from a patient with microphthalmia caused by an R200Q mutation in the VSX2 homeodomain region. No differences were noted between (R200Q)VSX2 and sibling control hiPSCs prior to OV generation. Thereafter, (R200Q)VSX2 hiPSC‐OVs displayed a significant growth deficit compared to control hiPSC‐OVs, as well as increased production of retinal pigmented epithelium at the expense of NR cell derivatives. Furthermore, (R200Q)VSX2 hiPSC‐OVs failed to produce bipolar cells, a distinctive feature previously observed in Vsx2 mutant mice. (R200Q)VSX2 hiPSC‐OVs also demonstrated delayed photoreceptor maturation, which could be overcome via exogenous expression of wild‐type VSX2 at early stages of retinal differentiation. Finally, RNAseq analysis on isolated hiPSC‐OVs implicated key transcription factors and extracellular signaling pathways as potential downstream effectors of VSX2‐mediated gene regulation. Our results establish hiPSC‐OVs as versatile model systems to study retinal development at stages not previously accessible in humans and support the bona fide nature of hiPSC‐OV‐derived retinal progeny. Stem Cells 2014;32:1480–1492

Modeling Human Retinal Development with Patient-Specific Induced Pluripotent Stem Cells Reveals Multiple Roles for Visual System Homeobox 2: Modeling Retinogenesis with Human iPS Cells

Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate along the retinal lineage in a manner that mimics normal mammalian development. Under certain culture conditions, hiPSCs form optic vesicle‐like structures (OVs), which contain proliferating progenitors capable of yielding all neural retina (NR) cell types over time. Such observations imply conserved roles for regulators of retinogenesis in hiPSC‐derived cultures and the developing embryo. However, whether and to what extent this assumption holds true has remained largely uninvestigated. We examined the role of a key NR transcription factor, visual system homeobox 2 (VSX2), using hiPSCs derived from a patient with microphthalmia caused by an R200Q mutation in the VSX2 homeodomain region. No differences were noted between (R200Q)VSX2 and sibling control hiPSCs prior to OV generation. Thereafter, (R200Q)VSX2 hiPSC‐OVs displayed a significant growth deficit compared to control hiPSC‐OVs, as well as increased production of retinal pigmented epithelium at the expense of NR cell derivatives. Furthermore, (R200Q)VSX2 hiPSC‐OVs failed to produce bipolar cells, a distinctive feature previously observed in Vsx2 mutant mice. (R200Q)VSX2 hiPSC‐OVs also demonstrated delayed photoreceptor maturation, which could be overcome via exogenous expression of wild‐type VSX2 at early stages of retinal differentiation. Finally, RNAseq analysis on isolated hiPSC‐OVs implicated key transcription factors and extracellular signaling pathways as potential downstream effectors of VSX2‐mediated gene regulation. Our results establish hiPSC‐OVs as versatile model systems to study retinal development at stages not previously accessible in humans and support the bona fide nature of hiPSC‐OV‐derived retinal progeny. Stem Cells 2014;32:1480–1492