In Utero Restoration of Hindbrain Herniation in Fetal Myelomeningocele as Part of Prenatal Regenerative Therapy Program at Mayo Clinic.

OBJECTIVE: To assess our initial experience with prenatal restoration of hindbrain herniation following in utero repair of myelomeningocele (MMC). PATIENTS AND METHODS: Three consecutive patients with prenatally diagnosed MMC (between January 1, 2018 and September 30, 2018) were managed with open in utero surgery. As per institutional review board approval and following a protocol designed at the Mayo Clinic Maternal & Fetal Center, fetal intervention was offered between 19 0/7 and 25 6/7 weeks of gestation. Prenatal improvement of hindbrain herniation was the declared restorative end point. Obstetrical and perinatal outcomes were also assessed. RESULTS: Diagnosis of MMC was confirmed upon referral between 20 and 21 weeks' gestation by using fetal ultrasound and magnetic resonance imaging. In all cases reported here, the spinal defect was lumbosacral with evidence of hindbrain herniation. Open in utero MMC repair was performed between 24 and 25 weeks' gestation with no notable perioperative complications. Postprocedure fetal magnetic resonance imaging performed 6 weeks after in utero repair documented improvement of hindbrain herniation. Deliveries were at 37 weeks by cesarean section without complications. Most recent postnatal follow-ups were unremarkable at both 11 months (baby 1) and 3 months of age (baby 2), with mild ventriculomegaly. Antenatal and postnatal follow-up of baby 3 at 1 month of age was also unremarkable. CONCLUSION: Our study highlights the prenatal restoration of hindbrain herniation following in utero MMC repair in all cases presented here as an example of a prenatal regenerative therapy program in our institution.

Produção da proteína recombinante humana TGF-ß1 (fator do crescimento transformante beta 1) em células de mamífero / Production of recombinant human protein TGF-ß1 (Transforming Growth Factor Beta 1) in mammalian cells

O fator de crescimento transformante beta tipo 1, TGF-ß1, é uma proteína extracelular homodimérica secretada por vários tipos celulares, que pode ter ação parácrina ou endócrina. Essa proteína está envolvida em processos celulares de diferenciação, proliferação, mobilidade e formação de matriz extracelular. Além disso, é parte importante dos processos de regeneração tecidual, atuando, de maneira decisiva, no reparo, atraindo macrófagos e fibroblastos para o local da injúria e estimulando a angiogênese. Assim, considerando o papel desse peptídeo no processo regenerativo, o uso de TGF-ß1 como proteína terapêutica na área de Bioengenharia Tecidual é bastante promissor. Apesar disso, a venda dessa proteína, para fins terapêuticos, é inexistente no mercado e a proteína recombinante vendida, que só pode ser utilizada em pesquisas científicas, não é produzida nacionalmente e chega a custar R$200.000,00/mg. Nesse contexto, o objetivo do presente trabalho é desenvolver uma metodologia de produção do fator recombinante TGF-ß1 em células de ovário de hamster chinês (CHO), visando à obtenção de níveis altos de rendimento, e, futuramente, a transferência da tecnologia de produção para a iniciativa privada, tornando possível seu uso na Medicina Regenerativa, sozinho ou em combinação com outros fatores de crescimento. O cDNA de TGF-ß1 foi amplificado a partir de um banco de cDNA humano e clonado no vetor proprietário pNU1 de expressão de mamífero. A construção pNU1/TGF-ß1 foi utilizada para transfectar estavelmente células CHO DG44 e uma estratégia de co-amplificação foi utilizada para selecionar células transfectantes com maior número de cópias da sequência correspondente a TGF-ß1. Estas culturas foram submetidas ao processo de amplificação gênica com concentrações crescentes de metotrexato. Ensaios de Western Blot e ELISA foram realizados utilizando-se o meio condicionado pelas populações selecionadas e por clones superprodutores. Entre os 41clones obtidos, cinco apresentaram maiores níveis de produção de TGF-ß1, entre 1.000 e 2.000 ng/mL. Estes clones foram selecionados para a realização de testes de atividade in vitro utilizando-se células A549, que permitem avaliar a transição epitélio-mesênquima. Um ensaio de cicatrização de feridas em peles do dorso de camundongos foi padronizado e utilizado para avaliar a atividade in vivo do clone que apresentou melhor resultado in vitro. A proteína TGF-ß1 foi parcialmente purificada por HPLC em uma coluna de afinidade. Portanto, a proteína TGF-ß1 humana recombinante foi produzida, apresentando atividade biológica in vitro e in vivo, sendo capaz de reparar eficientemente feridas cutâneas. Essa iniciativa pode oferecer aos pacientes uma alternativa para o tratamento de lesões teciduais, acelerando a cicatrização de feridas e o reparo de tecidos

The transforming growth factor beta 1, TGF-ß1, is a homodimeric extracellular protein secreted by several cell types, which may have paracrine or endocrine action. This protein is involved in cellular processes of differentiation, proliferation, mobility and formation of extracellular matrix. In addition, it is an important part of the tissue regeneration processes, acting decisively on repair, attracting macrophages and fibroblasts to the site of injury and stimulating angiogenesis. Therefore, considering the role of this peptide in the regenerative process and the use of TGF-ß1 as a therapeutic protein in the field of Tissue Bioengineering is very promising. Despite this, the sale of this protein for therapeutic purposes is nonexistent in the market and the recombinant protein available in the market, which can only be used in scientific research, is not produced nationally and the costs are in the order of R$ 200,000.00/mg. In this context, the objective of the present work is to develop a methodology for the production of the TGF-ß1 recombinant factor in Chinese hamster ovary (CHO) cells, aiming at obtaining high yields, and, in the future, transfering the production technology to the private initiative, allowing its use in Regenerative Medicine, alone or in combination with other growth factors. The TGF-ß1 cDNA was amplified from a human cDNA library and cloned into the proprietary pNU1 mammalian expression vector. The pNU1/TGF-ß1 construct was used to stably transfect CHO DG44 cells, and a co-amplification strategy was used to select transfectant cells with the largest number of gene copies. These cultures were subjected to the process of gene amplification with methotrexate. Western Blot and ELISA were used to assay the conditioned medium obtained from the selected cell populations and from overproducing cell clones. Among the 41 clones obtained, five presented higher levels of TGF-ß1 production, between 1,000 and 2,000 ng/mL. These clones were selected for in vitro activity testing using A549 cells to evaluate the epithelial-mesenchymal transition. Awound healing assay on mouse dorsal skin was standardized and used to evaluate the in vivo activity of the cell clone which displayed the highest result in vitro. The TGF-ß1 protein was partially purified by HPLC on an affinity column. Therefore, the recombinant human TGF-ß1 protein was produced and shown to display biological activity both in vitro and in vivo, being able to eficiently repair cutaneous wounds. This initiative may provide patients with an alternative treatment for tissue damage, accelerating wound healing and tissue repair

Perspectiva histórica de la plaqueta y su conexión con la enfermedad tromboembólica y su tratamiento (pasado, presente y futuro) / Historical perspective of the platelet and its connection to thromboembolic disease and its treatment (past, present and future)

En este artículo se hace una revisión histórica, desde la mitad del siglo pasado hasta la actualidad, de la importancia de la plaqueta y de su actividad funcional en relación con la enfermedad tromboembólica. Esta revisión se inicia en la primera mitad del siglo pasado, cuando este «elemento forme» de la sangre era conocido, casi en exclusiva, por su participación en la hemostasia primaria. Sin embargo, en las décadas de los años ochenta y noventa, se convierte en un actor clave y central en la formación del trombo arterial para ser considerado, en la actualidad y los próximos años, en un factor importante, pero uno más, en la génesis y desarrollo de los procesos trombóticos. Se hace un repaso de los descubrimientos clave que han permitido esta evolución y como ello ha hecho posible el descubrimiento y desarrollo de fármacos capaces de inhibir la agregación plaquetaria, en particular el triflusal, un antiagregante plaquetario fruto de la investigación llevada a cabo en el Laboratorio Uriach. Es por este descubrimiento que la historia de la i+d en Uriach ha estado también unida, durante más de 4 décadas, a la historia evolutiva de la plaqueta y de la enfermedad tromboembólica (AU)

This article gives a historical review, from the middle of the last century to the present, of the importance of the platelet and its functional activity in thromboembolic disease. This review begins in the first half of the last century when this blood component was almost exclusively known for its role in primary haemostasis. However, in the nineteen-eighties and nineties, it becomes a key player in the formation of arterial thrombus. Currently and in the coming years it will be considered as an important factor, but just one more, in the formation and development of thrombotic processes. A review is done of the key events that have permitted this evolution and how this has enabled the discovery and development of platelet inhibitor drugs, in particular Trifusal, an anti-platelet agent obtained from research carried out by Uriach Laboratories. Due to this discovery, R&d history at Uriach has been linked, for over four decades, to the historical evolution of both the platelet and thromboembolic disease

Culture, maintenance and control of mouse embryonic stem cells

Embryonic stem cells are considered to have a potential use in RegenerativeMedicine due to three key properties: a) pluripotency, thatis, the plasticity to differentiate into any cell type through asymmetricdivisions, b) high proliferation rate and clonal regeneration throughsymmetric divisions, and c) the ability, although limited, to maintainan undifferentiated state in culture. For these reasons, the use ofmouse embryonic stem cells is a fundamental tool for the decipheringof the operating mechanisms that need to occur for the in vitrodifferentiation of insulin-producing cells. This strategy may have anenormous implication in Regenerative Medicine for the treatment ofdiabetes. In this context, the in vitro culture of embryonic stem cellsrequires the control of a number of factors. Variability in such factorscan compromise the differentiation potential of these cells in bothdirected and spontaneous differentiation protocols, resulting in a dramaticalteration of the final cell product

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