Manufacturing Parameters for the Creation of Clinical-Grade Human-Induced Pluripotent Stem Cell Lines From Umbilical Cord Mesenchymal Stromal Cells.

Induced pluripotent stem cells (iPSCs) are reprogrammed cells with a remarkable capacity for unlimited expansion and differentiation into various cell types. Companies worldwide are actively engaged in developing clinical-grade iPSC lines to address the needs of regenerative medicine, immunotherapies, and precision medicine. However, ensuring the safety and quality of iPSCs is essential, with adherence to Good Manufacturing Practices (GMP) and ethical considerations being paramount. Perinatal cell and tissue banks, such as umbilical cord (UC) blood and tissue banks, are emerging as ideal sources for generating iPSCs due to their unique characteristics and GMP compliance. These banks provide access to immature cells with limited environmental exposure, known family and medical histories of donors, and readily available resources, thereby reducing the time and cost associated with personalized treatment strategies. This study describes the establishment of the first clinical-grade iPSC lines from umbilical cord mesenchymal stromal cells in Brazil. The process involved rigorous quality control measures, safety assessments, and adherence to regulatory standards, resulting in iPSCs with the necessary characteristics for clinical use, including sterility, genomic integrity, and stability. Importantly, the study contributes to the development of a Current Good Manufacturing Practice-compliant iPSC production pipeline in Brazil, using commercially available, chemically defined, and xeno-free products, along with validation by national outsourced laboratories, thereby facilitating the adoption of this technology within the country. The study emphasizes Brazil's contribution to the progress of translational medicine and the promotion of scientific advancements within the field of regenerative and precision medicine.

CD73 mitigates ZEB1 expression in papillary thyroid carcinoma.

BACKGROUND: ZEB1, a core transcription factor involved in epithelial-mesenchymal transition (EMT), is associated with aggressive cancer cell behavior, treatment resistance, and poor prognosis across various tumor types. Similarly, the expression and activity of CD73, an ectonucleotidase implicated in adenosine generation, is an important marker of tumor malignancy. Growing evidence suggests that EMT and the adenosinergic pathway are intricately linked and play a pivotal role in cancer development. Therefore, this study focuses on exploring the correlations between CD73 and ZEB1, considering their impact on tumor progression. METHODS: We employed CRISPR/Cas9 technology to silence CD73 expression in cell lines derived from papillary thyroid carcinoma. These same cells underwent lentiviral transduction of a reporter of ZEB1 non-coding RNA regulation. We conducted studies on cell migration using scratch assays and analyses of cellular speed and polarity. Additionally, we examined ZEB1 reporter expression through flow cytometry and immunocytochemistry, complemented by Western blot analysis for protein quantification. For further insights, we applied gene signatures representing different EMT states in an RNA-seq expression analysis of papillary thyroid carcinoma samples from The Cancer Genome Atlas. RESULTS: Silencing CD73 expression led to a reduction in ZEB1 non-coding RNA regulation reporter expression in a papillary thyroid carcinoma-derived cell line. Additionally, it also mitigated ZEB1 protein expression. Moreover, the expression of CD73 and ZEB1 was correlated with alterations in cell morphology characteristics crucial for cell migration, promoting an increase in cell polarity index and cell migration speed. RNA-seq analysis revealed higher expression of NT5E (CD73) in samples with BRAF mutations, accompanied by a prevalence of partial-EMT/hybrid state signature expression. CONCLUSIONS: Collectively, our findings suggest an association between CD73 expression and/or activity and the post-transcriptional regulation of ZEB1 by non-coding RNA, indicating a reduction in its absence. Further investigations are warranted to elucidate the relationship between CD73 and ZEB1, with the potential for targeting them as therapeutic alternatives for cancer treatment in the near future.

Adenosine metabolism by mesenchymal stromal cells isolated from different human tissues.

Mesenchymal stromal cells (MSCs) have unique biological properties and play important functions, which make them attractive tools for cell-based therapies. The basic mechanisms of these cells are not fully understood. However, the adenosinergic pathway contributes to the main effects attributed to MSCs. Adenosine is a highly immunosuppressive molecule and exerts a central role in inflammation by neutralizing the proinflammatory ATP influence. This nucleoside is produced by purinergic signaling, an important physiological pathway for MSCs, which involves proliferation, migration, differentiation, and apoptosis. Therefore, in this study, we analyzed the extracellular AMP hydrolysis and consequent adenosine production, as well as the expression of CD73 and adenosine receptors on the cell surface of MSCs isolated from different human tissues: dermis (D-MSCs), adipose tissue (AD-MSCs), and umbilical cord (UC-MSCs). All cells confirmed their multipotent capacity by adipogenic, osteogenic, and chondrogenic differentiation, as well as the expression of cell surface markers including CD44 + , CD105 + , and CD90 + . All MSCs expressed similar levels of CD73 and CD26 without a statistical difference among the different tissues, whereas ADA expression was lower in AD-MSCs. In addition, A1R and A3R mRNA levels were higher in D-MSCs and AD-MSCs, respectively. Enzymatic assay showed that AD-MSCs have the highest hydrolysis rate of AMP, leading to increased amount of adenosine production. Moreover, despite all MSCs completely hydrolyze extracellular AMP generating adenosine, the pattern of nucleosides metabolism was different. Therefore, although MSCs share certain characteristics as the multilineage potential and immunophenotype, they show different adenosinergic profiles according to tissue origin.

A Systematic Review of the Role of Purinergic Signalling Pathway in the Treatment of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global health concern. Three years since its origin, despite the approval of vaccines and specific treatments against this new coronavirus, there are still high rates of infection, hospitalization, and mortality in some countries. COVID-19 is characterised by a high inflammatory state and coagulation disturbances that may be linked to purinergic signalling molecules such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine (ADO), and purinergic receptors (P1 and P2). These nucleotides/nucleosides play important roles in cellular processes, such as immunomodulation, blood clot formation, and vasodilation, which are affected during SARS-CoV-2 infection. Therefore, drugs targeting this purinergic pathway, currently used for other pathologies, are being evaluated in preclinical and clinical trials for COVID-19. In this review, we focus on the potential of these drugs to control the release, degradation, and reuptake of these extracellular nucleotides and nucleosides to treat COVID-19. Drugs targeting the P1 receptors could have therapeutic efficacy due to their capacity to modulate the cytokine storm and the immune response. Those acting in P2X7, which is linked to NLRP3 inflammasome activation, are also valuable candidates as they can reduce the release of pro-inflammatory cytokines. However, according to the available preclinical and clinical data, the most promising medications to be used for COVID-19 treatment are those that modulate platelets behaviour and blood coagulation factors, mainly through the P2Y12 receptor.

A novel method to pack cryopreserved amniotic membrane for wound dressing-the pathway through validation of a new biological product.

Amniotic membrane (AM) has been widely used as a biological dressing for many pathologies and illnesses worldwide, and products derived from this tissue have been commercially available in several countries. In Brazil, regulatory agencies have recently authorized its clinical use as a non-experimental therapy for burns, diabetic and venous stasis ulcers, and intrauterine adhesions. In this study, we present our pathway through validating the first available service in the country of AM cryopreservation, with a protocol for long-term storage in high-efficiency nitrogen cryogenic freezers and a specific way of packing the tissue for optimal clinical handling and efficient storage space utilization while preserving live cells and the tissue's biological properties. Using gauze as support, cryoprotectant dimethyl sulfoxide and product presentation as a multilayer roll exhibited the best cell viability results and maintained the tissue integrity and presence of stem/progenitor cells. Essential proteins involved in tissue regeneration and immune and antimicrobial control were detected from the secretome of cryopreserved tissue similar to fresh tissue. Furthermore, immunogenic markers, such as human leukocyte antigens, were detected at very low levels in the tissue, confirming their low immunogenicity. Finally, we demonstrate that the tissue can be kept under refrigerated conditions for up to 7 d for further use, maintaining sterility and considerable cell viability. Our cryopreservation and storage protocol kept the AM viable for at least 20 months. In conclusion, this study enabled us to determine a novel efficient protocol for long-term AM preservation for future clinical applications.

Relocating to a new facility: The challenge of a cord blood banking transferral in Brazil.

BACKGROUND: Our cord blood banking facility planned and executed a transferral of its entire operation to a new site in the South of Brazil. Transporting LN2 freezers is a complex process in which extensive planning is essential to minimize the risks of damaging products or storage units. METHODS: To fulfill this objective, we constructed a detailed relocation plan consisting of four phases and risk mitigation measures, collaborated with the representatives of all departments, regulating agencies, and professionals from the transport company, and assembled a validation plan for cryogenic freezers and the viability of cord blood units. RESULTS: The new facility was prepared in accordance with the project plan, local legislation, quality system program requirements, and accreditation agency guidance. A 12-h operation of moving the cryogenic freezers was conducted successfully, with no loss or damage of client samples or equipment. CONCLUSION: Through the development and execution of a transferral plan, the engagement of appropriate partners, and compliance with security measures from health and government agencies, a successful transferral of a cord blood banking facility operation in its entirety can be successfully accomplished.

The crossroads of adenosinergic pathway and epithelial-mesenchymal plasticity in cancer.

Epithelial-mesenchymal transition (EMT) is a key mechanism related to tumor progression, invasion, metastasis, resistance to therapy and poor prognosis in several types of cancer. However, targeting EMT or partial-EMT, as well as the molecules involved in this process, has remained a challenge. Recently, the CD73 enzyme, which hydrolyzes AMP to produce adenosine (ADO), has been linked to the EMT process. This relationship is not only due to the production of the immunosuppressant ADO but also to its role as a receptor for extracellular matrix proteins, being involved in cell adhesion and migration. This article reviews the crosstalk between the adenosinergic pathway and the EMT program and the impact of this interrelation on cancer development and progression. An in silico analysis of RNAseq datasets showed that several tumor types have a significant correlation between an EMT score and NT5E (CD73) and ENTPD1 (CD39) expressions, with the strongest correlations being in prostate adenocarcinoma. Furthermore, it is evident that the cooperation between EMT and the adenosinergic pathway in tumor progression is context and tumor-dependent. The increased knowledge about this topic will help broaden the view to explore new treatments and therapies for different types of cancer.

Rat Adipose-Derived Stromal Cells (ADSCs) Increases the Glioblastoma Growth and Decreases the Animal Survival.

Many studies have shown that mesenchymal stromal cells (MSCs) and their secreted factors may modulate the biology of tumor cells. However, how these interactions happen in vivo remains unclear. In the present study, we investigated the effects of rat adipose-derived stromal cells (ADSCs) and their conditioned medium (ADSC-CM) in glioma tumor growth and malignancy in vivo. Our results showed that when we co-injected C6 cells plus ADSCs into the rat brains, the tumors generated were larger and the animals exhibited shorter survival, when compared with tumors of the animals that received only C6 cells or C6 cells pre-treated with ADSC-CM. We further showed that the animals that received C6 plus ADSC did not present enhanced expression of CD73 (a gene highly expressed in ADSCs), indicating that the tumor volume observed in these animals was not a mere consequence of the higher density of cells administered in this group. Finally, we showed that the animals that received C6 + ADSC presented tumors with larger necrosis areas and greater infiltration of immune cells. These results indicate that the immunoregulatory properties of ADSCs and its contribution to tumor stroma can support tumor growth leading to larger zones of necrosis, recruitment of immune cells, thus facilitating tumor progression. Our data provide new insights into the way by which ADSCs and tumor cells interact and highlight the importance of understanding the fate and roles of MSCs in tumor sites in vivo, as well as their intricate crosstalk with cancer cells.

Imatinib mesylate affects extracellular ATP catabolism and expression of NTPDases in a chronic myeloid leukemia cell line.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence of the t(9;22)(q34;q11) translocation. First-line therapy for CML consists of treatment with imatinib mesylate, which selectively inhibits the BCR-ABL protein by competing for its ATP-binding site. Adenine nucleotide signaling is modulated by the ectonucleotidases and this pathway is related to tumorigenic processes. Considering the relationship between ATP and cancer, we aimed to evaluate the influence of imatinib mesylate on the expressions and functions of the NTPDase and ecto-5'-nucleotidase (CD73) enzymes in imatinib-sensitive and -resistant K-562 cell lines. mRNA analysis showed that K-562 cells express all ENTPDs and NT5E. However, when treated with imatinib mesylate for 24 h, the expression of ENTPD1, -2, -3 and -5 increased, leading to a higher nucleotides hydrolysis rate. HPLC analysis identified increased ATP degradation in cells after 24 h of treatment, with consequent ADP and AMP formation, corroborating the increase in gene and protein expression of ectonucleotidases as observed in previous results. On the other hand, we observed that imatinib-resistant K-562 cells presented a decrease in nucleotide hydrolysis and expressions of ENTPD1 and -5. These results suggest an involvement of imatinib in modulating ectonucleotidases in CML that will need further investigation. Since these ectonucleotidases have important catalytic activities in the tumor microenvironment, their modulation in CML cells may represent an important therapeutic approach to regulate levels of extracellular adenine nucleotides.

Antitumor action of diphenyl diselenide nanocapsules: In vitro assessments and preclinical evidence in an animal model of glioblastoma multiforme.

BACKGROUND: Gliomas are the most aggressive malignant tumors of the central nervous system. The diphenyl diselenide [(PhSe)2] is an organoselenium compound that has multiple pharmacological properties. Previous reports showed that (PhSe)2 nanoencapsulation potentiates its in vitro antitumoral action and reduces its toxicity. OBJECTIVE: In this sense, the current study was designed to further evaluate the (PhSe)2 antitumoral effect by a set of in vitro techniques using a glioma cell line as well as by an animal model of gliobastoma. METHODS: For the in vitro tests, the cell viability, propidium iodide uptake and nitrite levels of rat glioma C6 cells were determined after incubation with free (PhSe)2 or (PhSe)2-loaded nanocapsules (NC). The glioblastoma model was induced by implantation of C6 glioma cells in the right striatum of rats. Following, animals were submitted to a repeated intragastric administration treatment with (PhSe)2 or NC (PhSe)2 (1 mg/kg/day for 15 days) to assess the possible antitumor effect. MAIN FINDINGS: Both compound forms decreased the C6 glioma cells viability without causing any effect in astrocytes cells (healthy control). Importantly, the NC (PhSe)2 had superior cytotoxic effect than its free form and increased the nitrite content. Independent of the (PhSe)2 forms, the intragastric treatment reduced brain tumor size and caused neither alteration in the plasma renal and hepatic markers of function nor in the parameters of oxidative balance in brain, liver and kidneys. PRINCIPAL CONCLUSIONS: The (PhSe)2 nanoencapsulation improved its cytotoxic effect against C6 glioma cells and both compound forms attenuated the tumor development.

Characterization of soluble CD39 (SolCD39/NTPDase1) from PiggyBac nonviral system as a tool to control the nucleotides level.

Extracellular ATP (eATP) and its metabolites have emerged as key modulators of different diseases and comprise a complex pathway called purinergic signaling. An increased number of tools have been developed to study the role of nucleotides and nucleosides in cell proliferation and migration, influence on the immune system and tumor progression. These tools include receptor agonists/antagonists, engineered ectonucleotidases, interference RNAs and ectonucleotidase inhibitors that allow the control and quantification of nucleotide levels. NTPDase1 (also called apyrase, ecto-ATPase and CD39) is one of the main enzymes responsible for the hydrolysis of eATP, and purified enzymes, such as apyrase purified from potato, or engineered as soluble CD39 (SolCD39), have been widely used in in vitro and in vivo experiments. However, the commercial apyrase had its effects recently questioned and SolCD39 exhibits limitations, such as short half-life and need of high doses to reach the expected enzymatic activity. Therefore, this study investigated a non-viral method to improve the overexpression of SolCD39 and evaluated its impact on other enzymes of the purinergic system. Our data demonstrated that PiggyBac transposon system proved to be a fast and efficient method to generate cells stably expressing SolCD39, producing high amounts of the enzyme from a limited number of cells and with high hydrolytic activity. In addition, the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other enzymes from the purinergic system. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.

Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105+ , CD44+ , CD14- , CD34- , CD45- , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc.