Full text
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| Author(s): |
Alexander Rodrigo Ferreira
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2019-10-04 |
| Examining board members: |
Dimas Tadeu Covas
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| Advisor: | Dimas Tadeu Covas |
| Abstract | |
There are several models seeking to reproduce normal and inefficient erythropoiesis in vitro. One of the biggest challenges lies in the development of adult-like erythroid cells with functional activity. The effectiveness of this differentiation can be monitored by the evaluation of morphological, immunophenotypic and gene and protein expression changes. Hemoglobins are globin chain tetramers directly related to the functional activity of the mature erythroid cell, functioning as important biomarkers of terminal erythroid differentiation. Throughout human development, different globins are expressed with distinct oxygen affinities. Initially, there is an expression predominance of embryonic globins, which are replaced by fetal and later by adults. This process is called globin switching and its regulation is not fully understood. The present study proposed to generate and characterize the in vitro hematopoietic / erythroid progenitors\' differentiation from induced pluripotency stem cells (iPSC), K562 lineage and CD34+ progenitors and evaluate their globin switching. This process was monitored by morphological, immunophenotypic and gene expression analyzes. The differentiation of iPSC was performed using the embryoid body formation protocol and resulted in decreased expression of pluripotency markers and increase of CD34+, CD36+, CD71+, and CD235a+ and globins gene expression, however they were not able to form colonies in the methylcellulose assay. There was an increase in the of Band 3 positive cells frequency and an increment in epsilon globin gene expression during the K562 strain differentiation. The differentiation of CD34+ progenitors was efficient in generating terminally differentiated erythroid cells, mimicking the erythropoiesis stages. An increased CD36+, CD235a+, and Band 3+ expression was observed. There was an important change in the globins gene expression and on the switching regulators, being an important model for the study of this regulation process. In addition, a targeted proteomics method was developed for monitoring the globins expression profile that can be applied in in vitro erythroid differentiation protocols. (AU) | |
| FAPESP's process: | 17/08057-2 - The globin switching of the in vitro hematopoietic differentiation |
| Grantee: | Alexander Rodrigo Ferreira |
| Support Opportunities: | Scholarships in Brazil - Master |