Xiufeng Zhong

Identification of tumorigenic retinal stem-like cells in human solid retinoblastomas.

Retinoblastoma (RB) is the most common malignant tumor of the retina in human children. Although it has been hypothesized for a long time that RB derives from multipotent retinal stem cells (RSCs) or retinoblasts, the direct evidence that the presence of tumorigenic RSCs in RB tumors is still lacking. Some studies indicate that malignant tumors contain tumor stem cells similar to their normal tissue stem cell counterparts. With in vitro culture and differentiation method we demonstrate that tumorigenic retinal stem‐like cells (RSLCs) indeed exist in RB lesions and that RB tumor‐derived cultures encompass undifferentiated cells capable of extensive proliferation as clonal nonadherent neurospheres and can differentiate into different retinal cells in vitro. Interestingly, cultured cells expressed retinal development related genes including nestin, CD133, pax6, chx10 and Rx, and overexpressed Bmi‐1, a gene required for self‐renewal and proliferation of stem cells. Significantly, when these cultured cells were intraocularly transplanted into SCID mice, they gave rise to new tumors with histomorphological features and immunophenotypes similar to their parental primary RBs. The results show that RBs contain tumorigenic RSLCs that contribute to tumorigenesis. This study provides a new insight to investigate the histogenesis of RBs and establishes a model for other RB research.

Role of MEF feeder cells in direct reprogramming of mousetail-tip fibroblasts

Pluripotent stem cells can be induced from somatic cells by the transcription factors Oct3/4, Sox2, c‐Myc and Klf4 when co‐cultured with mouse embryonic fibroblast (MEF) feeder cells. To date, the role of the feeder cells in the reprogramming process remains unclear. In this study, using a comparative analysis, we demonstrated that MEF feeder cells did not accelerate reprogramming or increase the frequency of induced pluripotent stem (iPS) cell colonies. However, feeder conditions did improve the growth of primary iPS colonies and were necessary for passaging the primary colonies after reprogramming was achieved. We further developed a feeder‐free culture system for supporting iPS growth and sustaining pluripotency by adding bFGF and activin A (bFA) to the medium. These data will facilitate the generation of human iPS cells without animal feeders for regenerative medicine.

HiPSC-derived retinal ganglion cells grow dendritic arbors and functional axons on a tissue-engineered scaffold

Numerous therapeutic procedures in modern medical research rely on the use of tissue engineering for the treatment of retinal diseases. However, the cell source and the transplantation method are still a limitation. Previously, it was reported that a self-organizing three-dimensional neural retina can be induced from human-induced pluripotent stem cells (hiPSCs). In this study, we disclose the generation of retinal ganglion cells (RGCs) from the neural retina and their seeding on a biodegradable poly (lactic-co-glycolic acid) (PLGA) scaffold to create an engineered RGC-scaffold biomaterial. Moreover, we explored the dendritic arbor, branching point, functional axon and action potential of the biomaterial. Finally, the cell-scaffold was transplanted into the intraocular environment of rabbits and rhesus monkeys. STATEMENT OF SIGNIFICANCE As a part of the mammalian central nervous system (CNS), the retinal ganglion cell (RGC) shows little regenerative capacity. With the use of medical biomaterial for cells seeding and deliver, a new domain is now emerging that uses tissue engineering therapy for retinal disease. However, previous studies utilized RGCs from rodent model, which has limitations for human disease treatment. In the present study, we generated RGCs from hiPSCs-3D neural retina and then seeded these RGCs on PLGA scaffold to create an engineered RGC-scaffold biomaterial. Moreover, we assessed the transplantation method for biomaterial in vivo. Our study provides a technique to produce the engineered human RGC-scaffold biomaterial.

p16INK4a expression in retinoblastoma: a marker of differentiation grade

BackgroundThe tumor suppressor protein p16INK4a has been extensively studied in many tumors with very different results, ranging from its loss to its clear overexpression, which may be associated with degree of tumor differentiation and prognosis. However, its expression remains unclear in human retinoblastoma (RB), a common malignant tumor of retina in childhood. The aim of this study was to explore the expression pattern of p16INK4a in RB, and the correlation between p16INK4a expression and histopathological features of RB.MethodsSixty-five cases of RB were retrospectively analyzed. Paraffin-embedded blocks were retrieved from the archives of ocular pathology department at Zhongshan Ophthalmic Center of Sun Yat-sen University, China. Serial sections were cut and subjected to hematoxylin and eosin staining. Immunohistochemical staining was further done with antibodies p16INK4a, CRX and Ki67. The correlation of p16 INK4a expression with CRX and Ki67 and clinicopathological features of RB were analyzed.ResultsRB tumor histologically consists of various differentiation components including undifferentiated (UD) cells, Homer-Wright rosettes (HWR) or Flexner-Winterstein rosettes (FWR) and fleurettes characteristic of photoreceptor differentiation or Retinocytoma (RC). p16INK4a expression was negative in both fleurette region and the residual retinal tissue adjacent to the tumor, weakly to moderately positive in FWR, strongly positive in both HWR and UD region. However, CRX had the reverse expression patterns in comparison with p16INK4a. It was strongly positive in photoreceptor cells within the residual retina and fleurettes, but weakly to moderately positive in UD area. Together with Ki67 staining, high p16INK4a expression was associated with poor histological differentiation of RB tumors, which had higher risk features with the optic nerve invasion and uveal invasion.Conclusionsp16INK4a expression increased with the decreasing level of cell differentiation of RBs. RB tumors extensively expressing p16INK4a tended to have higher risk features with poor prognosis. This study suggested that p16INK4a would be a valuable molecular marker of RB to distinguish its histological phenotypes and to serve as a predictor of its prognosis.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_180

Derivation and Identification of Motor Neurons from Human Urine-Derived Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have provided new opportunities for motor neuron disease (MND) modeling, drug screening, and cellular therapeutic development. Among the various types of iPSCs, urine-derived iPSCs have become a promising source of stem cells because they can be safely and noninvasively isolated and easily reprogrammed. Here, for the first time, we differentiated urine-derived iPSCs (urine-iPSCs) into motor neurons (MNs) and compared the capacity of urine-iPSCs and cord-blood-derived iPSCs (B-iPSCs) to differentiate into MNs. With the use of small molecules, mature MNs were generated from urine-iPSCs as early as 26 days in culture. Furthermore, in coculture with muscle cells, MNs projected long axons and formed neuromuscular junctions (NMJs). Immunofluorescence and PCR confirmed the expression levels of both MN and NMJ markers. The comparison of the ratios of positive labeling for MN markers between urine-iPSCs and B-iPSCs demonstrated that the differentiation potentials of these cells were not significantly different. The abovementioned results indicate that urine-iPSCs are a new, promising source of stem cells for MND modeling and further cellular therapeutic development.

An Optimized System for Effective Derivation of Three‐Dimensional Retinal Tissue Via Wnt Signaling Regulation

Effective derivation of three‐dimensional (3D) retinal tissue from human‐induced pluripotent stem cells (hiPSCs) could provide models for drug screening and facilitate patient‐specific retinal cell replacement therapy. However, some hiPSC lines cannot undergo 3D self‐organization and show inadequate differentiation efficiency to meet clinical demand. In this study, we developed an optimized system for derivation of 3D retinal tissue. We found that the Wnt signaling pathway antagonist Dickkopf‐related protein 1 (DKK‐1) rescued the inability of differentiated retinal progenitors to self‐organize. By evaluating DKK‐1 expression and supplying DKK‐1 if necessary, retinal organoids were differentiated from six hiPSC lines, which were reprogramed from three common initiating cell types. Retinal tissues derived from the optimized system were well organized and capable of surviving for further maturation. Thus, using this system, we generated retinal tissues from various hiPSC lines with high efficiency. This novel system has many potential applications in regenerative therapy and precision medicine. Stem Cells 2018;36:1709–1722

A potential model for studying the plasticity and reprogramming of human epidermal stem cells through preimplantation blastocyst microinjection

Microinjection of adult stem cells (ASCs) into blastocysts provides a classic model for studying ASC plasticity. To explore the molecular mechanisms that govern the reprogramming of ASCs, we evaluated the experimental model through microinjection of human epidermal stem cells (hEpiSCs) into mouse blastocysts. Mouse blastocysts underwent regular embryogenesis after microinjection of allogeneic cells, confirmed by morphological observation and embryo cell counting. hEpiSCs survive and integrate into mouse embryos, by monitoring the migration of injected cells at 2, 4, 12, 16 and 24 h. In this xenogeneic system, hEpiSCs could be reprogrammed within 24 h, as evidenced by the silencing of CK15 and Integrinβ1 gene expression, without activation of Oct4 and Nanog. Microinjection of hEpiSCs into mouse blastocysts provides an efficient model for studying the molecular mechanisms of their plasticity. Moreover, the possibility of inducing pluripotent stem cells without transgenes or viruses can be entertained.

Generation of Retinal Organoids with Mature Rods and Cones from Urine-Derived Human Induced Pluripotent Stem Cells

Urine cells, a body trash, have been successfully reprogrammed into human induced pluripotent stem cells (U-hiPSCs) which hold a huge promise in regenerative medicine. However, it is unknown whether or to what extent U-hiPSCs can generate retinal cells so far. With a modified retinal differentiation protocol without addition of retinoic acid (RA), our study revealed that U-hiPSCs were able to differentiate towards retinal fates and form 3D retinal organoids containing laminated neural retina with all retinal cell types located in proper layer as in vivo. More importantly, U-hiPSCs generated highly mature photoreceptors with all subtypes, even red/green cone-rich photoreceptors. Our data indicated that a supplement of RA to culture medium was not necessary for maturation and specification of U-hiPSC-derived photoreceptors at least in the niche of retinal organoids. The success of retinal differentiation with U-hiPSCs provides many opportunities in cell therapy, disease modeling, and drug screening, especially in personalized medicine of retinal diseases since urine cells can be noninvasively collected from patients and their relatives.