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Study question: Is there a possibility of reseeding cancer cells potentially present in frozen ovarian tissue from patients with central nervous system (CNS) tumours? Summary answer: Malignancy reseeding in cryopreserved ovarian tissue from 20 patients with CNS tumours was not detected by histology, immunohistochemistry (IHC), molecular biology or xenotransplantation. What is known already: Ovarian metastasis potential has been documented in patients with leukaemia, borderline ovarian tumours, advanced breast cancer and Ewing sarcoma. However, data on the safety of transplanting frozen-thawed ovarian tissue from cancer patients with CNS tumours are still lacking. Study design, size, duration: This prospective experimental study was conducted in an academic gynaecology research laboratory using cryopreserved ovarian cortex from 20 patients suffering from CNS tumours. Long-term (5 months) xenografting was performed in immunodeficient mice. Participants/materials, setting, methods: Subjects enrolled in the study were suffering from one of six types of CNS tumours including medulloblastoma, ependymoma, primitive neuroectodermal tumours, astrocytoma, glioblastoma and germinoma. The presence of malignant cells was investigated with disease-specific markers for each patient in cryopreserved and xenografted ovarian tissue by histology, IHC via expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP), and reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) for quantification of GFAP and ENO2 gene amplification. Main results and the role of chance: Serial sections of cryopreserved and xenografted ovarian tissue from 20 patients showed no malignant cells by histology. All samples were negative for NSE and GFAP, although these neural markers were expressed extensively in the patients' primary tumours. Analysis by RT-ddPCR revealed no cancer cells detected in cryopreserved and xenografted ovarian fragments from subjects with a

2. Study question: Is cryopreserved ovarian tissue transplantation safe in patients with central nervous system (CNS) tumors? 3. Summary answer: Cancer cell contamination was not detected in any ovarian samples from patients with CNS tumors by histological analysis, immunohistochemistry, molecular biology or long-term xenotransplantation.

Is there a possibility of reseeding cancer cells potentially present in frozen ovarian tissue from patients with central nervous system (CNS) tumours?, info:eu-repo/semantics/published

Genetic alterations in genes lead to peroxisome biogenesis disorder. In humans, they are associated with Zellweger spectrum disorders (ZSD). No validated treatment has been shown to modify the dismal natural history of ZSD. Liver transplantation (LT) improved clinical and biochemical outcomes in mild ZSD patients. Hepatocyte transplantation (HT), developed to overcome LT limitations, was performed in a mild ZSD 4-year-old child with encouraging short-term results. Here, we evaluated low dose (12.5 million hepatocytes/kg) and high dose (50 million hepatocytes/kg) syngeneic male HT via intrasplenic infusion in the -G844D NMRI mouse model which recapitulates a mild ZSD phenotype. HT was feasible and safe in growth retarded ZSD mice. Clinical (weight and food intake) and biochemical parameters (very long-chain fatty acids, abnormal bile acids, etc.) were in accordance with ZSD phenotype but they were not robustly modified by HT. As expected, one third of the infused cells were detected in the liver 24 h post-HT. No liver nor spleen microchimerism was detected after 7, 14 and 30 days. Future optimizations are required to improve hepatocyte engraftment in -G844D NMRI mouse liver. The mouse model exhibited the robustness required for ZSD liver-targeted therapies evaluation.

One of the main challenges in liver cell therapy (LCT) is the induction of a tolerogenic microenvironment to promote graft acceptance in the recipient. Little is known about the immunomodulatory potential of the hepatic cells used in liver cell therapy. In this work, we wanted to evaluate the immunosuppressive properties of human hepatocytes and adult-derived human liver stem/progenitor cells (ADHLSCs), as well as the potential involvement of the immunomodulatory molecule HLA-G. We demonstrated that both cell types were capable of inhibiting the proliferative response of PBMCs to an allogenic stimulus and that the immune inhibitory potential of ADHLSCs, although lower than that of hepatocytes, increased after hepatogenic differentiation. We demonstrated that liver cells express HLA-G and that the immune inhibition pattern was clearly associated to its expression. Interestingly, HLA-G expression increased after the third step of differentiation, wherein oncostatin M (OSM) was added. A 48 hr treatment with OSM was sufficient to induce HLA-G expression in ADHLSCs and result in immune inhibition. Surprisingly, blocking HLA-G partially reversed the immune inhibition mediated by hepatocytes and differentiated ADHLSCs, but not that of undifferentiated ADHLSCs, suggesting that additional immune inhibitory mechanisms may be used by these cells. In conclusion, we demonstrated that both hepatocytes and ADHLSCs present immunomodulatory properties mediated, at least in part, through HLA-G, which can be upregulated following hepatogenic differentiation or liver cell pretreatment with OSM. These observations open up new perspectives for the induction of tolerance following LCT and for potential therapeutic applications of these liver cells.

BACKGROUND. Cell transplantation is in clinical development for the treatment of various ailments including acquired and inborn hepatic diseases. Detection and quantification of the donor cells after infusion remain difficult. Traditional methods (sex-based FISH, HLA mismatch, and Short Tandem Repeat PCR) can only achieve low levels of sensitivity (1%) and therefore are seldom used. The use of a droplet digital PCR (ddPCR) assay based on mismatch of null alleles is a promising alternative. METHODS. We selected genes with a high frequency of null genotype in the general population (SRY, RHD, TRY6, LEC3C, GSTM1, and GSTT1) and investigated their expression by liver progenitor cell donors and liver cell therapy recipients, in order to identify genes of interest for each donor/recipient couple. We first validated the detection of microchimerism by ddPCR and then used these assays to detect and quantify microchimerism in pre- and postinfusion liver biopsies. RESULTS. We validated the ddPCR detection of the selected genes based on linearity, precision, lack of inhibition, and accuracy, and we established limits of blank, limits of detection, and limits of quantification to ensure the reliability of the results. After genotyping donors and recipients, we were able to identify at least one gene of interest for each donor/recipient couple. We detected donor cells in the three patients posttransplantation. However, analysis of several biopsies taken at the same timepoint revealed a heterogeneous cell distribution. In addition, the values obtained remained below the limit of quantification. Therefore, the actual quantification of microchimerism may not be entirely accurate. CONCLUSIONS Overall, our study demonstrates that the detection of microchimerism post-liver cell transplantation can be performed using ddPCR amplification of null allele genes expressed by the donor but absent from the recipient. However, this technique can be extended to other cell types and target organs

One of the main challenges in liver cell therapy (LCT) is the induction of a tolerogenic microenvironment to promote graft acceptance in the recipient. Little is known about the immunomodulatory potential of the hepatic cells used in liver cell therapy. In this work, we wanted to evaluate the immunosuppressive properties of human hepatocytes and adult-derived human liver stem/progenitor cells (ADHLSCs), as well as the potential involvement of the immunomodulatory molecule HLA-G. We demonstrated that both cell types were capable of inhibiting the proliferative response of PBMCs to an allogenic stimulus and that the immune inhibitory potential of ADHLSCs, although lower than that of hepatocytes, increased after hepatogenic differentiation. We demonstrated that liver cells express HLA-G and that the immune inhibition pattern was clearly associated to its expression. Interestingly, HLA-G expression increased after the third step of differentiation, wherein oncostatin M (OSM) was added. A 48 hr treatment with OSM was sufficient to induce HLA-G expression in ADHLSCs and result in immune inhibition. Surprisingly, blocking HLA-G partially reversed the immune inhibition mediated by hepatocytes and differentiated ADHLSCs, but not that of undifferentiated ADHLSCs, suggesting that additional immune inhibitory mechanisms may be used by these cells. In conclusion, we demonstrated that both hepatocytes and ADHLSCs present immunomodulatory properties mediated, at least in part, through HLA-G, which can be upregulated following hepatogenic differentiation or liver cell pretreatment with OSM. These observations open up new perspectives for the induction of tolerance following LCT and for potential therapeutic applications of these liver cells., SCOPUS: ar.j, info:eu-repo/semantics/published

Adult-derived human liver stem/progenitor cells (ADHLSCs) have the potential to alleviate liver injury. However, the optimal delivery route and long-term biodistribution of ADHLSCs remains unclear. In this paper, we used a triple fusion reporter system to determine the kinetic differences in the biodistribution of ADHLSCs following intrasplenic (IS) and intrahepatic (IH) administration in SCID-beige mice. ADHLSCs were transduced with a lentiviral vector expressing a triple-fusion reporter comprising renilla luciferase, monomeric red fluorescent protein and truncated HSV-1 thymidine kinase. The stability and duration of the transgenes, and the effects of transduction on the cell properties were evaluated in vitro. The acute retention and long-term engraftment in vivo were revealed by positron emission tomography (PET) and bioluminescence imaging (BLI), respectively, followed by histochemical analysis. We showed that ADHLSCs can be safely transduced with the triple fusion reporter. Radiolabeled ADHLSCs showed acute cell retention at the sites of injection. The IH group showed a confined BLI signal at the injection site, while the IS group displayed a dispersed distribution at the upper abdominal liver area, and a more intense signal. In conclusion, ADHLSCs could be monitored by BLI for up to 4 weeks with a spread out biodistribution following IS injection. .

Introduction Adult-derived human liver stem cells (ADHLSCs) are currently in clinical development for the treatment of Urea Cycle Disorders (UCD). Clinical and preclinical data seem to indicate a higher clinical effect than what could be expected from the number of cells that have engrafted, suggesting that other mechanisms may be at play. We have previously demonstrated that ADHLSCs produce Extracellular Vesicles, EVs (MP, microparticles; and EXO, exosomes), which have been shown to mediate intracellular communication in other systems by delivering proteins, lipids and/or genetic information (coding and non-coding RNAs) to recipient cells. Therefore, the aim of this study was to determine the precise role of EVs in ADHLSC-mediated correction of UCD. Methods ADHLSCs were cultured for 2 days in DMEM supplemented with 10% EXO-free FBS and 1% P/S. The conditioned medium was collected, and MP and EXO fractions were harvested by serial ultracentrifugation. Transmission electron microscopy (TEM), western blotting and nanoparticle tracking analysis were used to evaluate the presence, purity and abundance of MP and EXO. RNA from EVs was stained with SytoRNA, which only fluoresces upon integration into RNA, to investigate RNA transfer from EVs to rat hepatocytes. Droplet digital PCR (ddPCR) was performed on RNA extracted from the MP and EXO as well as rat hepatocytes previously incubated with EVs to investigate the presence of human mRNAs of interest. Results We confirmed that ADHLSCs produce both MP and EXO. Characterization of the mRNA by ddPCR showed expression of ASL, ASS, and CPS1 in EVs, mainly in MPs. SytoRNA staining of the EV RNA allowed us to show transfer of EV RNA to over 60% of rat hepatocytes in vitro. Finally, we demonstrated transfer of human mRNAs of interest from EVs to rat hepatocytes using ddPCR. Summary/Conclusion In summary, our study shows that ADHLSC-derived EVS contain mRNA encoding for some of the deficient enzymes in UCD and are capable of transfer

BACKGROUND: With the exception of liver transplantation, there is no cure for hemophilia, which is currently managed by preemptive replacement therapy. Liver-derived stem cells are in clinical development for inborn and acquired liver diseases and could represent a curative treatment for hemophilia A. The liver is a major factor VIII (FVIII) synthesis site, and mesenchymal stem cells have been shown to control joint bleeding in animal models of hemophilia. Adult-derived human liver stem cells (ADHLSCs) have mesenchymal characteristics and have been shown able to engraft in and repopulate both animal and human livers. Thus, the objectives were to evaluate the potency of ADHLSCs to control bleeding in a hemophilia A patient and assess the biodistribution of the cells after intravenous injection. METHODS: A patient suffering from hemophilia A was injected with repeated doses of ADHLSCs via a peripheral vein (35 million In-oxine-labeled cells, followed by 125 million cells the next day, and 3 infusions of 250 million cells every 2 weeks thereafter; total infusion period, 50 days). RESULTS: After cell therapy, we found a temporary (15 weeks) decrease in the patient's FVIII requirements and severe bleeding complications, despite a lack of increase in circulating FVIII. The cells were safely administered to the patient via a peripheral vein. Biodistribution analysis revealed an initial temporary entrapment of the cells in the lungs, followed by homing to the liver and to a joint afflicted with hemarthrosis. CONCLUSION: These results suggest the potential use of ADHLSCs in the treatment of hemophilia A.

Introduction Zellweger spectrum disorders (ZSD) are peroxisome biogenesis disorders (PBD) attributable to mutations in PEX genes that lead to metabolites accumulation (phytanic acid, …). Supportive management is currently considered the standard of care, since no treatment has showed clinical benefits. Adult-Derived Human Liver Stem Cells (ADHLSCs) are isolated from the parenchymal fraction of human livers not suitable for transplantation. In contrast to hepatocytes, they resist well to cryopreservation and they are able to proliferate in vitro making them suitable for cell therapy. Clinical trials for urea cycle disorders and Crigler-Najjar syndrome are ongoing with ADHLSCs. Aim The goal of our study was to determine whether ADHLSCs would be a suitable candidate for the cell therapy treatment of ZSD. Methods We characterized peroxisome biogenesis and function in ADHLSCs isolated from healthy donors and a ZSD patient (PEX1 mutation) used as negative control. Peroxisome biogenesis was evaluated by immunofluorescence staining against PMP70, a peroxisome membrane protein, and catalase, a peroxisome matrix protein. We further transfected cells with a plasmid coding for a green fluorescent protein linked to a peroxisome targeting signal (GFP-SKL) to assess their capacity to import peroxisomal proteins. Finally, peroxisome function was evaluated by a phytanic acid degradation assay. Results Immunocytochemistry staining showed a granular colocalized pattern for PMP70 and catalase in ADHLSCs isolated from healthy donors. ADHLSCs from the ZSD patient exhibited a coarse granular pattern for PMP70 and absence of catalase staining (peroxisomal ghosts) meaning a peroxisome biogenesis disorder. GFP-SKL transfection lead to a punctiform or a diffuse green staining pattern in healthy ADHLSCs or peroxisome biogenesis deficient ADHLSCs, respectively. Healthy ADHLSCs degraded phytanic acid at 24.72 ± 7.88 pmol/min*mg protein, a speed comparable to previous studies on hepatocytes by Wa

Adult-derived human liver stem/progenitor cells (ADHLSCs) have the potential to alleviate liver injury. However, the optimal delivery route and long-term biodistribution of ADHLSCs remain unclear. In this article, we used a triple fusion reporter system to determine the kinetic differences in the biodistribution of ADHLSCs following intrasplenic (IS) and intrahepatic (IH) administration in severe combined immunodeficiency/beige mice. ADHLSCs were transduced with a lentiviral vector expressing a triple fusion reporter comprising renilla luciferase, monomeric red fluorescent protein, and truncated HSV-1 thymidine kinase. The stability and duration of the transgenes, and the effects of transduction on the cell properties were evaluated in vitro. The acute retention and long-term engraftment in vivo were revealed by positron emission tomography and bioluminescence imaging (BLI), respectively, followed by histochemical analysis. We showed that ADHLSCs can be safely transduced with the triple fusion reporter. Radiolabeled ADHLSCs showed acute cell retention at the sites of injection. The IH group showed a confined BLI signal at the injection site, while the IS group displayed a dispersed distribution at the upper abdominal liver area, and a more intense signal. In conclusion, ADHLSCs could be monitored by BLI for up to 4 weeks with a spread out biodistribution following IS injection., SCOPUS: ar.j, info:eu-repo/semantics/published

Background and aims Extracellular vesicles, EVs (MP, microparticles; and EXO, exosomes) are nano-size vesicles released by many cell types. They mediate intracellular communication by delivering proteins, lipids and/or genetic information (coding and non-coding RNAs) to recipient cells. Adult-derived human liver stem cells (ADHLSCs) are currently in clinical development for the treatment of liver diseases. Clinical and preclinical data seem to indicate a higher clinical effect than what could be expected from the number of cells that have engrafted, suggesting that other mechanisms may be at play. Therefore, we aim to know whether the EVs have therapeutic effects and whether they can contribute to ADHLSC-mediated correction of Crigler Najjar syndrome. Methods ADHLSCs were cultured for 2 days in DMEM supplemented with 10% EXO free FBS and 1% P/S. The conditioned medium was collected, and MP and EXO fractions were harvested by serial centrifugation. FACS and western blotting were used to evaluate the presence of MP and EXO using specific markers. RT-qPCR was performed on RNA extracted from the MP and EXO to investigate the presence of mRNAs of interest in each sample. Results We demonstrated that CD40L, a specific marker of MP, is expressed in ADHLSC-derived MP, while ALIX and CD9, which are specific EXO markers, are expressed in ADHLSC-derived EXO. ALIX was also highly expressed in the EXO fraction derived from a metabolic donor suffering from Crigler-Najjar syndrome, showing that this “EXO marker” can be found in EXO from both healthy and metabolic sources. In order to analyze the EXO fraction by flow cytometry, EXO were incubated with EXO-specific beads and stained for CD9. Using this method, we were able to confirm the expression of CD9 at the surface of the EXO. Characterization of the mRNA by qPCR showed a high expression of UGT1A1 in EXO. This finding supports the hypothesis that EXO may play a role in ADHLSC-mediated correction of Crigler-Najjar syndrome. Conc

Mesenchymal stromal cells (MSCs) are known to have potential therapeutic benefits for a number of diseases. However, many studies report low engraftment levels, regardless of the target organ. One possible explanation could be that MSCs do not express the necessary receptors for engraftment. Indeed, MSCs appear to use a similar mechanism to leukocytes to engraft into injured organs, relying on various receptors for rolling, firm adhesion, and transmigration. In this study, we conducted an extensive surface molecule screening of adult-derived human liver stem/progenitor cells (ADHLSC) in an attempt to shed some light on this subject. We observed that ADHLSCs lack expression of most of the costimulatory molecules tested. Furthermore, study of the adhesion molecule profile of ADHLSCs revealed that they do not express selectin ligands or LFA-1 which are, respectively, involved in the rolling process and the firm adhesion. In addition, ADHLSCs slightly express VLA-4 and lose expression of CXCR4 altogether on their surface during culture expansion. However, ADHLSCs express all the integrin couples and matrix metalloproteinases needed to bind and integrate the extracellular matrix once the endothelial barrier is crossed. Collectively, these results suggest that binding to the endothelium may be the critical weak point in the engraftment process.

Objectives & Study: Cell transplantation is a promising alternative to liver transplantation. The cells can be either hepatocytes or liver derived progenitors. In both cases tracking the cell fate after infusion and monitoring the chimerism is an important point. Traditional method (sex-based FISH, HLA mismatch, Short Tandem Repeat PCR) achieve only low level of sensitivity (1%) and are seldom used. The use of quantitative Real-Time PCR based on mismatch of null allele is a promising alternative. Methods: We selected four genes with a high level of null genotype in population (SRY, RHD, GSTM1, GSTT1). We evaluated the genotype distribution on a panel of 25 blood donor. We created an artificial chimerism based on DNA mix or liver derived progenitor to test sensitivity, accuracy and variability. We also tested the method on the biopsy of a patient which had cell progenitor infusion for G6PD deficiency. The biopsies were taken at 3.5 month and 7 month. Results: Analysis combining the four genes had sensitivity up to 0.01% of chimerism, with a good accuracy and below 3% of coefficient of variation for intra and interassay experiment. The informativeness is 57% for the four genes. The measured chimerism of the patient was 0.045% of the total liver mass, corresponding to 5% engraftment of the transplanted cells (SD : 0.02) for the right liver biopsy and 0.025% (SD : 0.013) for the left liver biopsy at 3.5 month. At 7 month the level of chimerism was under the limit of detection but present. It should be noted that the patient presented to acute enteritis with diarrhea at five month which could be have caused to a diminished immunosuppression. Conclusion: The chimerism determination with real-time PCR amplification of null allele is a reliable and sensitive tool. It could be easily implemented in follow up of patient with cell infusion for liver disease.