Xenogenic Application of Human Placenta-Derived Mesenchymal Stromal Cells in a Porcine Large Animal Model.

In animal models, cell therapies for different diseases or injuries have been very successful. Preclinical studies with cells aiming at a stroke, heart attack, and other emergency situations were promising but sometimes failed translation in clinical situations. We, therefore, investigated if human placenta-derived mesenchymal stromal cells can be injected in pigs without provoking rejection to serve as a xenogenic transplantation model to bridge preclinical animal studies to more promising future preclinical studies. Male human placenta-derived mesenchymal stromal cells were isolated, expanded, and characterized by flow cytometry, in vitro differentiation, and quantitative reverse-transcription polymerase chain reaction to prove their nature. Such cells were injected into the sphincter muscle of the urethrae of female pigs under visual control by cystoscopy employing a Williams needle. The animals were observed over 7 days of follow-up. Reactions of the host to the xenogeneic cells were explored by monitoring body temperature, and inflammatory markers including IL-1ß, CRP, and haptoglobin in blood. After sacrifice on day 7, infiltration of inflammatory cells in the tissue targeted was investigated by histology and immunofluorescence. DNA of injected human cells was detected by PCR. Upon injection in vascularized porcine tissue, human placenta-derived mesenchymal stromal cells were tolerated, and systemic inflammatory parameters were not elevated. DNA of injected cells was detected in situ 7 days after injection, and moderate local infiltration of inflammatory cells was observed. The therapeutic potential of human placenta-derived mesenchymal stromal cells can be explored in porcine large animal models of injury or disease. This seems a promising strategy to explore technologies for cell injections in infarcted hearts or small organs and tissues in therapeutically relevant amounts requiring large animal models to yield meaningful outcomes.

Regulation of Immune Checkpoint Antigen CD276 (B7-H3) on Human Placenta-Derived Mesenchymal Stromal Cells in GMP-Compliant Cell Culture Media.

Therapies utilizing autologous mesenchymal cell delivery are being investigated as anti-inflammatory and regenerative treatments for a broad spectrum of age-related diseases, as well as various chronic and acute pathological conditions. Easily available allogeneic full-term human placenta mesenchymal stromal cells (pMSCs) were used as a potential pro-regenerative, cell-based therapy in degenerative diseases, which could be applied also to elderly individuals. To explore the potential of allogeneic pMSCs transplantation for pro-regenerative applications, such cells were isolated from five different term-placentas, obtained from the dissected maternal, endometrial (mpMSCs), and fetal chorion tissues (fpMSCs), respectively. The proliferation rate of the cells in the culture, as well as their shape, in vitro differentiation potential, and the expression of mesenchymal lineage and stem cell markers, were investigated. Moreover, we studied the expression of immune checkpoint antigen CD276 as a possible modulation of the rejection of transplanted non-HLA-matched homologous or even xeno-transplanted pMSCs. The expression of the cell surface markers was also explored in parallel in the cryosections of the relevant intact placenta tissue samples. The expansion of pMSCs in a clinical-grade medium complemented with 5% human platelet lysate and 5% human serum induced a significant expression of CD276 when compared to mpMSCs expanded in a commercial medium. We suggest that the expansion of mpMSCs, especially in a medium containing platelet lysate, elevated the expression of the immune-regulatory cell surface marker CD276. This may contribute to the immune tolerance towards allogeneic pMSC transplantations in clinical situations and even in xenogenic animal models of human diseases. The endurance of the injected comparably young human-term pMSCs may promote prolonged effects in clinical applications employing non-HLA-matched allogeneic cell therapy for various degenerative disorders, especially in aged adults.

Laparoscopic Peritoneal Wash Cytology-Derived Primary Human Mesothelial Cells for In Vitro Cell Culture and Simulation of Human Peritoneum.

Peritoneal mucosa of mesothelial cells line the abdominal cavity, surround intestinal organs and the female reproductive organs and are responsible for immunological integrity, organ functionality and regeneration. Peritoneal diseases range from inflammation, adhesions, endometriosis, and cancer. Efficient technologies to isolate and cultivate healthy patient-derived mesothelial cells with maximal purity enable the generation of capable 2D and 3D as well as in vivo-like microfluidic cell culture models to investigate pathomechanisms and treatment strategies. Here, we describe a new and easily reproducible technique for the isolation and culture of primary human mesothelial cells from laparoscopic peritoneal wash cytology. We established a protocol containing multiple washing and centrifugation steps, followed by cell culture at the highest purity and over multiple passages. Isolated peritoneal mesothelial cells were characterized in detail, utilizing brightfield and immunofluorescence microscopy, flow cytometry as well as Raman microspectroscopy and multivariate data analysis. Thereby, cytokeratin expression enabled specific discrimination from primary peritoneal human fibroblasts. Raman microspectroscopy and imaging were used to study morphology and biochemical properties of primary mesothelial cell culture compared to cryo-fixed and cryo-sectioned peritoneal tissue.

Prostate cancer tissues with positive TMPRSS2-ERG-gene-fusion status may display enhanced nerve density.

Innervation of prostate cancer (CaP) tissue favors tumor progression and metastasis but the regulation of innervation in CaP is unclear. The oncogenic transcription factor ERG is commonly induced by a typical TMPRSS2-ERG (TE) gene fusion in CaP and may affect innervation. Here, we analyzed whether nerve density of CaP tissue is related to TE status or perineural infiltration status of CaP tissue. In parallel, we measured several members of the neuropilin/plexin/semaphorin family (NRP, PLXN, and SEMA) as possible targets mediating innervation. The TE-gene-fusion status was determined at the mRNA level in CaP tissues by nested RT-PCR. Transcript levels were analyzed by quantitative RT-PCR in CaP tissue or cell line homogenate. ERG was analyzed by immunostaining, and the nerve density was evaluated by immunostaining for PGP9.5 and axonal neurofilament. Data were analyzed by correlation (Spearman), linear regression, Mann-Whitney U test, and contingency table analyses. TE-positive (TE-1) vs. TE-negative (TE-0) CaP tissues displayed significantly enhanced ERG-mRNA levels (TE-0: -4.183; TE-1: -2.994, P < 0.001) and ERG immunostaining (Erg-IH score; TE-0: 0.4211; TE-1: 1.391; P < 0.0001). Notably, the nerve density was significantly increased in CaP tissue samples with positive TE status compared to negative TE status (TE-0, ND score = 1.5; TE-1, ND score = 2.0; P <0.01). NRP1, NRP2, PLXNA2, PLXNB1, SEMA3A, and SEMA4B mRNAs were detectable in CaP tissues and CaP cell lines at quite heterogeneous levels. In CaP tissues, we observed significant positive correlations of ERG with NRP2, PLXNA2, PLXNB1, and SEMA4B. TE-positive CaP tissues displayed enhanced nerve density. ERG correlated with some NRP/PLXN/SEMA components suggesting possible regulatory relevance of ERG for CaP innervation.

Assessing blood platelets as RNA biomarker source for prostate cancer.

CONTEXT: Blood platelets may offer as RNA biomarker source for cancer as recently described for an oncogenic transcript in glioma patients and for PCA3 in prostate cancer (PCa) patients. OBJECTIVE: Here, we elaborated on this aspect for PCa. MATERIALS AND METHODS: PCA3 and other PCa-associated RNA markers were measured in platelets of PCa patients (cases) and healthy subjects (controls) in comparison to PCa cell lines by relative quantitative RT-PCR. RESULTS: The RNA markers displayed heterogeneous expression patterns in cell lines and platelets, however, without significant differences between cases and controls. DISCUSSION AND CONCLUSION: The data do not support platelets as a profitable RNA source for early detection of PCa. Nonetheless, certain PCa-derived RNA markers in platelets may merit further investigation as potential prognostic biomarkers for PCa.