In vitro characterisation of foetal human neural progenitors, their astroglial derivates, and effects of released factors and extracellular matrix on axon regeneration

Human neural progenitor cells (hNPC) and human mesenchymal stromal cells (hMSC) have both been reported to be able to promote improved functional recovery after grafting into experimental spinal cord injuries. Transplantation of pre-differentiated neural progenitor cells has been shown to be an effective strategy to support repair of the spinal cord. In particular, the implantation of astrocyte restricted precursors may represent a means of promoting spinal cord recovery through the maintenance and support of axonal regeneration. Although it has been demonstrated by our group that foetal hNPC, pre-differentiated to type I astrocyte restricted precursors (hNP-AC), promote more extensive axon regeneration by adult rat dorsal root ganglia (DRG) neurons in vitro than non-differentiated hNPC or hMSC, the mechanisms of action to support neuritic outgrowth remained unclear. Therefore, hNPC were characterised before and after in vitro differentiation to hNP-AC to provide a clearer definition of the changes that the cells had undergone. Furthermore, possible contributions of released trophic factors (i.e. present in conditioned culture medium) or substrate-mediated mechanisms via the extracellular matrix (ECM) of hMSC and hNP-AC to support neuritic outgrowth were investigated. In the first part of this study, the in vitro immunocytochemical- and mRNA expression profile of selected marker gens in hNPC and in the differentiated hNP-AC was quantified. Furthermore, proliferative activity was estimated with a metabolic and a morphological assay, whereby the latter, by measuring the increase in volume over time, provided an indirect indicator of proliferation within intact neurospheres. The hNPC grew as neurospheres, which were immunoreactive for the stem/progenitor cell related markers nestin, SOX2 as well as musashi and expressed CD133- and nestin mRNA. They showed spontaneous differentiation capacity into MAP2ab+ and TuJ1+ neuronal phenotypes as well as S100b+, GFAP+ and vimentin+ astroglial precursors. Whereas the differentiated hNP-AC demonstrated a similar immunocytochemical expression profile for the stem/ progenitor cell related markers nestin, SOX2, musashi and expressed CD133- and nestin mRNA in similar proportions to the hNPC, the expression of astroglial markers increased. A significantly higher proportion of hNP-AC were immunoreactive for S100b, GFAP and CD44. Likewise, hNP-AC demonstrated significantly increased CD44- and GFAP mRNA expression than the non-differentiated hNPC. Estimation of proliferative activity of hNPC and hNP-AC showed a reduced doubling time (DT) in hNP-AC with a value of 5.1 days (morphological assay) and 3.2 days (metabolic assay), contrasting to hNPC prior to in vitro differentiation with a DT of 3.5 days (morphological assay) and 1.7 days (metabolic assay). In the second part of this study, the contributions of trophic factors present in cell-conditioned medium (produced by either hNP-AC or hMSC) and their ECM in supporting DRG axon regeneration were investigated in vitro. Conditioned media from both cell types strongly supported neurite outgrowth over a defined PLL/laminin substrate. The ECM produced by the two cell types, however, demonstrated contrasting effects: hNP-AC ECM promoted substantial axon regeneration, but hMSC ECM supported relatively little axon growth. The combined application of hNP-AC ECM and hMSC conditioned media induced the greatest degree of axon outgrowth. The combination of hMSC ECM and hNP-AC conditioned medium, however, did not result in any enhanced axon growth over the values determined using hMSC ECM and control growth medium. Thus, although hNP-AC conditioned medium demonstrated potent trophic support for DRG axon growth, its effect was not supported by signals derived from hMSC ECM. This raises interesting questions for future experiments regarding the cross-talk between the intracellular signalling cascades activated by diffusible trophic factors and those activated by ECM-related molecules

In vitro characterisation of foetal human neural progenitors, their astroglial derivates, and effects of released factors and extracellular matrix on axon regeneration

Human neural progenitor cells (hNPC) and human mesenchymal stromal cells (hMSC) have both been reported to be able to promote improved functional recovery after grafting into experimental spinal cord injuries. Transplantation of pre-differentiated neural progenitor cells has been shown to be an effective strategy to support repair of the spinal cord. In particular, the implantation of astrocyte restricted precursors may represent a means of promoting spinal cord recovery through the maintenance and support of axonal regeneration. Although it has been demonstrated by our group that foetal hNPC, pre-differentiated to type I astrocyte restricted precursors (hNP-AC), promote more extensive axon regeneration by adult rat dorsal root ganglia (DRG) neurons in vitro than non-differentiated hNPC or hMSC, the mechanisms of action to support neuritic outgrowth remained unclear. Therefore, hNPC were characterised before and after in vitro differentiation to hNP-AC to provide a clearer definition of the changes that the cells had undergone. Furthermore, possible contributions of released trophic factors (i.e. present in conditioned culture medium) or substrate-mediated mechanisms via the extracellular matrix (ECM) of hMSC and hNP-AC to support neuritic outgrowth were investigated. In the first part of this study, the in vitro immunocytochemical- and mRNA expression profile of selected marker gens in hNPC and in the differentiated hNP-AC was quantified. Furthermore, proliferative activity was estimated with a metabolic and a morphological assay, whereby the latter, by measuring the increase in volume over time, provided an indirect indicator of proliferation within intact neurospheres. The hNPC grew as neurospheres, which were immunoreactive for the stem/progenitor cell related markers nestin, SOX2 as well as musashi and expressed CD133- and nestin mRNA. They showed spontaneous differentiation capacity into MAP2ab+ and TuJ1+ neuronal phenotypes as well as S100b+, GFAP+ and vimentin+ astroglial precursors. Whereas the differentiated hNP-AC demonstrated a similar immunocytochemical expression profile for the stem/ progenitor cell related markers nestin, SOX2, musashi and expressed CD133- and nestin mRNA in similar proportions to the hNPC, the expression of astroglial markers increased. A significantly higher proportion of hNP-AC were immunoreactive for S100b, GFAP and CD44. Likewise, hNP-AC demonstrated significantly increased CD44- and GFAP mRNA expression than the non-differentiated hNPC. Estimation of proliferative activity of hNPC and hNP-AC showed a reduced doubling time (DT) in hNP-AC with a value of 5.1 days (morphological assay) and 3.2 days (metabolic assay), contrasting to hNPC prior to in vitro differentiation with a DT of 3.5 days (morphological assay) and 1.7 days (metabolic assay). In the second part of this study, the contributions of trophic factors present in cell-conditioned medium (produced by either hNP-AC or hMSC) and their ECM in supporting DRG axon regeneration were investigated in vitro. Conditioned media from both cell types strongly supported neurite outgrowth over a defined PLL/laminin substrate. The ECM produced by the two cell types, however, demonstrated contrasting effects: hNP-AC ECM promoted substantial axon regeneration, but hMSC ECM supported relatively little axon growth. The combined application of hNP-AC ECM and hMSC conditioned media induced the greatest degree of axon outgrowth. The combination of hMSC ECM and hNP-AC conditioned medium, however, did not result in any enhanced axon growth over the values determined using hMSC ECM and control growth medium. Thus, although hNP-AC conditioned medium demonstrated potent trophic support for DRG axon growth, its effect was not supported by signals derived from hMSC ECM. This raises interesting questions for future experiments regarding the cross-talk between the intracellular signalling cascades activated by diffusible trophic factors and those activated by ECM-related molecules

Detection of Merkel Cell Polyomavirus in Seborrheic Keratosis

Seborrheic keratosis (SK) is the most common benign cutaneous neoplasm. A subset shows increased p16 expression. Since SK shares several features with verruca vulgaris, e.g., increased p16 expression, human papillomaviruses (HPV) have been suggested as possible causal agents. However, a relevant association could not be established between HPV and SK. In the present study we aimed to investigate the presence of Merkel cell polyomavirus (MCPyV) in relation to p16 expression in SK. P16 expression was investigated using immunohistochemistry (IHC). Presence of MCPyV was assessed in 23 formalin-fixed paraffin-embedded tissue samples of SK by molecular techniques (i.e., PCR and FISH) and IHC. 16/23 SK showed strong to moderate p16 expression. 6/23 of SK were MCPyV positive by PCR which was confirmed by FISH. Of interest, two samples with strong FISH signals also showed MCPyV expression as tested by IHC. Samples with weaker signal intensity were negative in IHC. P16 expression was not associated with the presence of MCPyV. Concluding, the detection of MCPyV DNA by PCR and FISH in SK reflects the widespread prevalence of MCPyV in the skin. However, low detection rates exclude MCPyV as a major pathogenic factor in SK, most likely representing a coincidental infection. P16 IHC does not appear as useful adjunctive surrogate marker for the presence of MCPyV in SK

Refining the Diagnosis of Congenital Nephrotic Syndrome on Long-term Stored Tissue: c.1097G>A (p.(Arg366His)) WT1 Mutation Causing Denys Drash Syndrome

Contains fulltext : 168099.pdf (Publisher’s version ) (Open Access)Congenital nephrotic syndrome (CNS) caused by a mutation in the Wilms tumor 1 suppressor gene (WT1) is part of Denys Drash Syndrome or Frasier syndrome. In the framework of genetic counseling, the diagnosis of CNS can be refined with gene mutation studies on long-term stored formalin-fixed paraffin-embedded tissue from postmortem examination. We report a case of diffuse mesangial sclerosis with perinatal death caused by a de novo mutation in the WT1 gene in a girl with an XY-genotype. This is the first case of Denys Drash Syndrome with the uncommon missense c.1097G>A [p.(Arg366His)] mutation in the WT1 gene which has been diagnosed on long-term stored formalin-fixed paraffin-embedded tissue in 1993. This emphasizes the importance of retained and adequately stored tissue as a resource in the ongoing medical care and counseling

Development and verification of new monoclonal orthopedia homeobox (OTP) specific antibodies for pulmonary carcinoid diagnostics

Background: Orthopedia homeobox (OTP) has shown to be a useful prognostic marker to predict outcome in pulmonary carcinoids, which is also supported by the World Health Organization. However, the discontinuation of the initially used polyclonal antibody and absence of a reliable routinely applicable monoclonal OTP antibody hampers implementation in routine diagnostics. Here, new monoclonal antibodies directed against OTP were developed and verified on formalin-fixed paraffin-embedded tissue of pulmonary neuroendocrine tumors (NETs) for clinical diagnostics. Methods: OTP specific monoclonal antibodies were produced from mice immunised with a recombinant human OTP protein fragment. Enzyme-linked immunosorbent assay (ELISA) positive hybridomas were evaluated using immunohistochemistry (IHC). Following epitope-mapping and isotyping, purified monoclonal antibodies were validated for IHC in formalin-fixed paraffin-embedded tissues, the optimal dilution was determined, and results were cross validated with the OTP polyclonal antibody (HPA039365, Atlas Antibodies). Staining protocols were optimized on two automated staining platforms and performance was harmonized using a tissue microarray (TMA). Results: Two clones (CL11222 and CL11225) were selected for purified monoclonal antibody (mAb) production. Intratumor heterogeneity assessment revealed similar performance for both clones. While clone CL11225 displayed a unique epitope compared to those present in the polyclonal antibody, this clone performed most similar to the polyclonal antibody. Cross-platform assessment revealed an excellent agreement for clone CL11225 while clone CL11222 showed somewhat discordant results on Dako. Conclusions: New monoclonal OTP specific antibodies have been developed and verified on different automated immunohistochemical staining platforms. The OTP specific monoclonal antibodies showed excellent agreement with the often-used polyclonal antibody allowing application in routine diagnostics

Development and verification of new monoclonal orthopedia homeobox (OTP) specific antibodies for pulmonary carcinoid diagnostics

Background: Orthopedia homeobox (OTP) has shown to be a useful prognostic marker to predict outcome in pulmonary carcinoids, which is also supported by the World Health Organization. However, the discontinuation of the initially used polyclonal antibody and absence of a reliable routinely applicable monoclonal OTP antibody hampers implementation in routine diagnostics. Here, new monoclonal antibodies directed against OTP were developed and verified on formalin-fixed paraffin-embedded tissue of pulmonary neuroendocrine tumors (NETs) for clinical diagnostics.  Methods: OTP specific monoclonal antibodies were produced from mice immunised with a recombinant human OTP protein fragment. Enzyme-linked immunosorbent assay (ELISA) positive hybridomas were evaluated using immunohistochemistry (IHC). Following epitope-mapping and isotyping, purified monoclonal antibodies were validated for IHC in formalin-fixed paraffin-embedded tissues, the optimal dilution was determined, and results were cross validated with the OTP polyclonal antibody (HPA039365, Atlas Antibodies). Staining protocols were optimized on two automated staining platforms and performance was harmonized using a tissue microarray (TMA).  Results: Two clones (CL11222 and CL11225) were selected for purified monoclonal antibody (mAb) production. Intratumor heterogeneity assessment revealed similar performance for both clones. While clone CL11225 displayed a unique epitope compared to those present in the polyclonal antibody, this clone performed most similar to the polyclonal antibody. Cross-platform assessment revealed an excellent agreement for clone CL11225 while clone CL11222 showed somewhat discordant results on Dako.  Conclusions: New monoclonal OTP specific antibodies have been developed and verified on different automated immunohistochemical staining platforms. The OTP specific monoclonal antibodies showed excellent agreement with the often-used polyclonal antibody allowing application in routine diagnostics