ABSTRACT Introduction Sickle cell disease (SCD) is a monogenic disease and it is estimated that 300,000 infants are born annually with it. Most treatments available are only palliative, whereas the allogeneic hematopoietic stem cell transplantation offers the only potential cure for SCD. Objective Generation of human autologous cells, when coupled with induced pluripotent stem cell (iPSC) technology, is a promising approach for developing study models. In this study, we provide a simple and efficient model for generating hematopoietic cells using iPSCs derived from a sickle cell anemia patient and an inexpensive in-house-prepared medium. Method This study used iPSCs previously generated from peripheral blood mononuclear cells (PBMCs) from a patient with sickle cell anemia (iPSC_scd). Hematopoietic and erythroid differentiation was performed in two steps. Firstly, with the induction of hematopoietic differentiation through embryoid body formation, we evaluated the efficiency of two serum-free media; and secondly, the induction of hematopoietic stem/progenitor cells to erythroid progenitor cells was performed. Results The patient-specific cell line generated CD34+/CD45+ and CD45+/CD43+ hematopoietic stem/progenitor cells and erythroid progenitors, comprising CD36+, CD71+ and CD235a+ populations, as well as the formation of hematopoietic colonies, including erythroid colonies, in culture in a semi-solid medium. Conclusion In conjunction, our results described a simple serum-free platform to differentiate human the iPSCs into hematopoietic progenitor cells. This platform is an emerging application of iPSCs in vitro disease modeling, which can significantly improve the search for new pharmacological drugs for sickle cell disease.

ABSTRACT Chimeric antigen receptor T cells (CAR-T), especially against CD19 marker, present in lymphomas and acute B leukemia, enabled a revolution in the treatment of hematologic neoplastic diseases. The manufacture of CAR-T cells requires the adoption of GMP-compatible methods and it demands the collection of mononuclear cells from the patient (or from the donor), generally through the apheresis procedure, T cell selection, activation, transduction and expansion ex vivo, and finally storage, usually cryopreserved, until the moment of their use. An important aspect is the quality control testing of the final product, for example, the characterization of its identity and purity, tests to detect any contamination by microorganisms (bacteria, fungi, and mycoplasma) and its potency. The product thawing and intravenous infusion do not differ much from what is established for the hematopoietic progenitor cell product. After infusion, it is important to check for the presence and concentration of CAR-T cells in the patient’s peripheral blood, as well as to monitor their clinical impact, for instance, the occurrence of short-term, such as cytokine release syndrome and neurological complications, and long-term complications, which require patient follow-up for many years.

ABSTRACT The adherence to accreditation programs proves the institutions' voluntary effort to pursue the quality and safety of their products and services by meeting internationally accepted standards audited by experts in the field, external to the service. Meeting such standards often exceeds domestic legal requirements. However, service providers are not released from complying with the legal requirements, both local and international, pertinent to the field. Accreditation programs use the precepts of the quality management system to validate and standardize processes, monitor results through quality control, proficiency testing, and indicators, and perform risk management. For cellular therapy services, the assessing agencies available in our field are the AABB/ABHH (American Association of Blood Banks/Brazilian Association of Hematology, Hemotherapy and Cellular Therapy) and FACT-JACIE (Foundation for the Accreditation of Cellular Therapy-Joint Accreditation Committee, ISCT/EBMT). Both agencies require that the accredited organization meets all the standards defined in each program. Applying services also have to establish and comply with a quality management standard that demonstrates procedural interrelationship to ensure product and service quality. This paper aims to concisely outline the essential features of those two accreditation programs, along with a brief overview of the accreditation process under each of them.

O retrovírus linfotrópico de células T humanas tipo 1 é o agente etiológico da leucemia das células T do adulto e da paraparesia espástica tropical/mielopatia associada ao HTLV-1. O genoma proviral é composto por 9.032 pares de bases, contendo genes estruturais e regulatórios. A glicoproteína transmembrana gp 21 é codificada pelo gene estrutural env. O desenvolvimento de metodologias para a expressão heteróloga de proteínas, assim como a obtenção de uma linhagem celular que expresse a gp 21 recombinante constitutivamente são os principais objetivos do trabalho. O fragmento codificante da gp 21 foi amplificado por Nested-PCR e clonado no vetor pCR2.1-TOPO. Posteriormente, foi realizada a subclonagem no vetor de expressão pcDNA3.1+. A transfecção da linhagem celular de mamíferos HEK 293 foi realizada de maneira transitória e permanente. A produção da gp 21 recombinante foi confirmada por citometria de fluxo e a linhagem celular produtora será utilizada em ensaios de imunogenicidade.

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.