Yongping Li

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.

Generation of Human Epidermis-Derived Mesenchymal Stem Cell-like Pluripotent Cells (hEMSCPCs)

We isolated human epidermis-derived mesenchymal stem cell-like pluripotent cells (hEMSCPCs) and demonstrate efficient harvesting, maintenance in vitro for at least 30 passages, reprogramming into multiple phenotypes in vivo, and integration into adult host tissues after injection into the mouse blastocyst to create chimeras. Cell phenotype was examined by karyotyping, immunostaining, immunofluorescence, and flow cytometry. A nested PCR protocol using primers specific for human SRY genes was designed to detect hEMSCPC-derived cells in female chimeric mice. FISH was used to validate the results of nested PCR. Results indicated that hEMSCPCs were derived from epidermis but were distinct from epidermal cells; they resembled mesenchymal stem cells (MSCs) morphologically and expressed the main markers of MSCs. About half of all female offspring of mice implanted with embryos injected with hEMSCPCs at the blastocyst stage harbored the human Y chromosome and tissue-specific human protein, thereby demonstrating the transdifferentiation of hEMSCPCs.

CY15, a Malignant Histiocytic Tumor That Is Phenotypically Similar to Immature Dendritic Cells

The origin and pathogenesis of histiocytic malignancies and the biology of the tumor cells are poorly understood. We have isolated a murine histiocytic tumor cell line (CY15) from a BALB/c IFNgamma(-/-) mouse and characterized it in terms of phenotype and function. The morphology, as judged by electron microscopy, and the surface marker phenotype suggests that CY15 cells are similar to immature dendritic cells (CD11c (low), MHC II (low), CD11b(+), B7.1(+), B7.2(+), and CD40(+)). The cells form tumors in BALB/c mice and metastasize to spleen, liver, lung, kidney, and to a lesser extend to lymph nodes and bone marrow, as judged by the growth of green fluorescent protein transfected tumor cells in mice. CY15 cells are capable of actively taking up antigen (FITC-ovalbumin) and can stimulate T lymphocytes in an allogenic mixed lymphocyte reaction but less effectively than their normal counterparts (immature dendritic cells). They respond to interleukin 4 (IL-4) with up-regulation of CD11c. If stimulated with IFNgamma the cells up-regulate MHC II, CD40 B7.1, and B7.2. Lipopolysaccharide induces the cells to up-regulate B7.1 and B7.2 and to secrete tumor necrosis factor alpha and IL-12. Based on these data, CY15 is a dendritic cell-like tumor cell line and may serve as a transplantable tumor model for histiocytosis in humans.

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

SV40 large T antigen‐transformed human primary normal and cancerous mammary epithelial cells are phenotypically similar but can be distinguished in 3D culture with selection medium

Human normal mammary epithelial cells (NMECs) have 2 major in vitro growth restrictions, senescence and crisis. Cellular immortalization is considered a hallmark of malignancy. However, cancerous mammary epithelial cells (CMECs) that are thought to have passed growth barriers in vivo usually cannot be established long‐term in vitro. Here we show that CMECs deprived of their natural environment and grown in conventional complete medium behave similar to NMECs, e.g., they stop producing telomerase and become senescent. Like NMECs, CMECs are rescued by SV40 large T (LT) from senescence but not from crisis. The telomere length of both LT‐transformed NMEC (N‐LT) and CMEC (C‐LT) cells first shortens but later partially recovers after telomerase activation. Both cell types upregulate ErbB2 expression, acquire genetic changes, remain long‐term dependent on LT and ErbB2 and are nontumorigenic. Despite these similarities, N‐LT and C‐LT cells cultured in selection medium show different growth characteristics in 3D culture and in vivo tumorigenesis. Thus, CMECs are under a comparable in vitro selective pressure in conventional monolayer culture as NMECs despite their in vivo malignancy. This data demonstrate that most primary breast cancer cells are still unable to overcome the in vitro growth restrictions and suggest that the relationship of in vitro immortalization and in vivo carcinogenesis should be re‐evaluated.

Expression levels of autophagy related proteins and their prognostic significance in retinocytoma and retinoblastoma.

AIMnTo discuss the prognostic significant of autophagy related proteins (ARPs) in retinoblastoma (RB) and to find the molecular marker to distinguish retinocytoma (RC) and RB by investigating the different expression profiling of microtubule-associated protein light chain 3 (LC3B) and other ARPs in RC and RB.nnnMETHODSnSpecimens with retinocytoma region (RCR) or mainly composed with Flexner-Winterstein rosettes (FWR) were screen out from 219 paraffin-embedded RB samples and respectively taken as RCR group and FWR group. Others were taken as undifferentiated (UD) group. Immunochemistry (IHC) of LC3B and electronic microscopy was used to identify autophagy. The IHC scores of LC3B and other ARPs, such as Beclin, PTEN, p27, p16(INK4a), mTOR and BCL-2 were compared and correlation analysis was applied to find potential proteins which may involve in autophagy regulation. The prognostics significance of LC3B was evaluated by comparing the high risk features (HRFs) in 3 groups of total 219 samples.nnnRESULTSnTwenty-one specimens with RCR and 36 specimens mainly composed with FWR were screen out. RCR cell had a high level of LC3B and lots of autophagic vacuoles. Beclin, PTEN, p27 had positive correlation with LC3, and p16(INK4a) had negative correlation, while the expression of mTOR and BCL-2 in RCR and RB region did not show any difference. Cases with RCR had lower rate of HRFs than undifferentiated cases.nnnCONCLUSIONnARPs had different expression pattern between RCR and other pathological types of RB, and could be ideal markers to distinguish RC from RB. Our finding indicated cases with RCR had favorable prognosis just like those with FWR.

Strengthening the role of ocular pathology in clinical thinking training targeting for house staff.

Ocular pathology serves as one vital branch subject of histopathology,and also as a basic ocular science analysing the pathogenesis of eye disease,the regular pattern of disease progress,and ocular morphology,tissue metabolism and functional changes noted during the onset of ocular diseases.