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1.
Cytotherapy ; 2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38944797

ABSTRACT

As the field of cell and gene therapy (CGT) continues to grow, so too must the infrastructure and regulatory guidance supporting the manufacture of these potentially life-saving products-especially early-phase products manufactured at an increasing number of academic or hospital-based facilities providing decentralized (or point of care) manufacturing. An important component of current good manufacturing practices, including those regulating cell and gene therapies, is the establishment of an effective environmental monitoring (EM) program. While several guidelines for establishing an EM program are available, these guidelines do not specifically address the unique aspects of manufacturing CGT products and they do not provide real-world evidence demonstrating the effectiveness of the program. Here, we describe the establishment and evolution of an EM program in a cell therapy manufacturing facility at an academic hospital. With 10 years of EM data, we analyze the effectiveness for identifying trends in environmental conditions and highlight important findings, with the aim of providing practical evidence and guidance for the development of future early-phase EM programs.

2.
J Immunol ; 202(8): 2372-2383, 2019 04 15.
Article in English | MEDLINE | ID: mdl-30833349

ABSTRACT

Decreased inflammation and cardiovascular mortality are evident in patients with end-stage chronic kidney disease treated by online hemodiafiltration. Extracellular vesicles (EV) are mediators of cell-to-cell communication and contain different RNA types. This study investigated whether mixed online hemodiafiltration (mOL-HDF) beneficial effects associate with changes in the RNA content of plasma EV in chronic kidney disease patients. Thirty bicarbonate hemodialysis (BHD) patients were randomized 1:1 to continue BHD or switch to mOL-HDF. Concentration, size, and microRNA content of plasma EV were evaluated for 9 mo; we then studied EV effects on inflammation, angiogenesis, and apoptosis of endothelial cells (HUVEC) and on osteoblast mineralization of vascular smooth muscle cells (VSMC). mOL-HDF treatment reduced different inflammatory markers, including circulating CRP, IL-6, and NGAL. All hemodialysis patients showed higher plasma levels of endothelial-derived EV than healthy subjects, with no significant differences between BHD and mOL-HDF. However, BHD-derived EV had an increased expression of the proatherogenic miR-223 with respect to healthy subjects or mOL-HDF. Compared with EV from healthy subjects, those from hemodialysis patients reduced angiogenesis and increased HUVEC apoptosis and VSMC calcification; however, all these detrimental effects were reduced with mOL-HDF with respect to BHD. Cell transfection with miR-223 mimic or antagomiR proved the role of this microRNA in EV-induced HUVEC and VSMC dysfunction. The switch from BHD to mOL-HDF significantly reduced systemic inflammation and miR-223 expression in plasma EV, thus improving HUVEC angiogenesis and reducing VSMC calcification.


Subject(s)
Endothelium, Vascular/immunology , Extracellular Vesicles , Gene Expression Regulation/immunology , Hemodiafiltration , MicroRNAs , Renal Insufficiency, Chronic , Uremia , Vascular Calcification , Adult , Aged , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Extracellular Vesicles/immunology , Extracellular Vesicles/metabolism , Female , Human Umbilical Vein Endothelial Cells , Humans , Inflammation/blood , Inflammation/immunology , Inflammation/pathology , Inflammation/therapy , Male , MicroRNAs/blood , MicroRNAs/immunology , Middle Aged , Renal Insufficiency, Chronic/blood , Renal Insufficiency, Chronic/immunology , Renal Insufficiency, Chronic/pathology , Renal Insufficiency, Chronic/therapy , Uremia/blood , Uremia/immunology , Uremia/pathology , Uremia/therapy , Vascular Calcification/blood , Vascular Calcification/immunology , Vascular Calcification/pathology , Vascular Calcification/therapy
3.
Int J Cancer ; 144(2): 322-333, 2019 01 15.
Article in English | MEDLINE | ID: mdl-30110127

ABSTRACT

Human liver stem-like cells (HLSC) and derived extracellular vesicles (EVs) were previously shown to exhibit anti-tumor activity. In our study, we investigated whether HLSC-derived EVs (HLSC-EVs) were able to inhibit tumor angiogenesis in vitro and in vivo, in comparison with EVs derived from mesenchymal stem cells (MSC-EVs). The results obtained indicated that HLSC-EVs, but not MSC-EVs, inhibited the angiogenic properties of tumor-derived endothelial cells (TEC) both in vitro and in vivo in a model of subcutaneous implantation in Matrigel. Treatment of TEC with HLSC-EVs led to the down-regulation of pro-angiogenic genes. Since HLSC-EVs carry a specific set of microRNAs (miRNAs) that could target these genes, we investigated their potential role by transfecting TEC with HLSC-EV specific miRNAs. We observed that four miRNAs, namely miR-15a, miR-181b, miR-320c and miR-874, significantly inhibited the angiogenic properties of TEC in vitro, and decreased the expression of some predicted target genes (ITGB3, FGF1, EPHB4 and PLAU). In parallel, TEC treated with HLSC-EVs significantly enhanced expression of miR-15a, miR-181b, miR-320c and miR-874 associated with the down-regulation of FGF1 and PLAU. In summary, HLSC-EVs possess an anti-tumorigenic effect, based on their ability to inhibit tumor angiogenesis.


Subject(s)
Extracellular Vesicles , Hepatocytes , Neovascularization, Pathologic , Stem Cells , Animals , Humans , Liver/cytology , Mice , Mice, SCID , Xenograft Model Antitumor Assays
4.
Stem Cell Rev Rep ; 15(1): 93-111, 2019 02.
Article in English | MEDLINE | ID: mdl-30191384

ABSTRACT

A potential therapeutic strategy for diabetes is the transplantation of induced-insulin secreting cells. Based on the common embryonic origin of liver and pancreas, we studied the potential of adult human liver stem-like cells (HLSC) to generate in vitro insulin-producing 3D spheroid structures (HLSC-ILS). HLSC-ILS were generated by a one-step protocol based on charge dependent aggregation of HLSC induced by protamine. 3D aggregation promoted the spontaneous differentiation into cells expressing insulin and several key markers of pancreatic ß cells. HLSC-ILS showed endocrine granules similar to those seen in human ß cells. In static and dynamic in vitro conditions, such structures produced C-peptide after stimulation with high glucose. HLSC-ILS significantly reduced hyperglycemia and restored a normo-glycemic profile when implanted in streptozotocin-diabetic SCID mice. Diabetic mice expressed human C-peptide and very low or undetectable levels of murine C-peptide. Hyperglycemia and a diabetic profile were restored after HLSC-ISL explant. The gene expression profile of in vitro generated HLSC-ILS showed a differentiation from HLSC profile and an endocrine commitment with the enhanced expression of several markers of ß cell differentiation. The comparative analysis of gene expression profiles after 2 and 4 weeks of in vivo implantation showed a further ß-cell differentiation, with a genetic profile still immature but closer to that of human islets. In conclusion, protamine-induced spheroid aggregation of HLSC triggers a spontaneous differentiation to an endocrine phenotype. Although the in vitro differentiated HLSC-ILS were immature, they responded to high glucose with insulin secretion and in vivo reversed hyperglycemia in diabetic SCID mice.


Subject(s)
Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/therapy , Hyperglycemia/complications , Hyperglycemia/therapy , Islets of Langerhans/physiology , Liver/cytology , Stem Cells/cytology , Adult , Animals , Biomarkers/metabolism , C-Peptide/metabolism , Cell Differentiation/drug effects , Gene Expression Regulation/drug effects , Glucose/pharmacology , Humans , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Islets of Langerhans/ultrastructure , Male , Mice, SCID , Phenotype , Protamines/pharmacology , Spheroids, Cellular/cytology , Spheroids, Cellular/drug effects , Stem Cells/drug effects
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