Evaluation of myelo-monocytic differentiation of congenital neutropenia patient derived induced pluripotent stem cells

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URI: http://hdl.handle.net/10900/131123
Dokumentart: PhDThesis
Date: 2022-08-22
Language: English
Faculty: 4 Medizinische Fakultät
Department: Medizin
Advisor: Skokowa, Julia (Professor Dr. Ph.D)
Day of Oral Examination: 2022-04-27
DDC Classifikation: 610 - Medicine and health
Other Keywords:
Induced pluripotent stem cell
severe congenital neutropenia
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Severe congenital neutropenia (CN) is a heterogenous group of disorders of the myeloid differentiation, defined by a maturation arrest of granulopoiesis at the promyelocyte/myelocyte stage and an absolute neutrophil count (ANC) of < 0,5x109/L. Furthermore, characteristic for CN patients are elevated monocyte counts. The reason for this shift in myelomonocytic differentiation is being studied in attempts for better understanding of myeloid decision making beyond the effects of the PU.1 to C/EBPα transcription factors expression ratio. We analyzed an index CN patient with an unusually strong shift from granulocytic to monocytic development and no mutations in the known CN- associated genes (e.g. HAX1, ELANE, G6PC3, JAGN1, SRP54). Next generation sequencing has revealed rare single nucleotide polymorphisms in the FLT3 and FLT1 genes. To evaluate the role of the V194M-FLT3 variant on the myelomonocytic development, we produced expression constructs with wild type and V194M mutated FLT3. We found the mutated FLT3 to be inadequately expressed on protein level. Furthermore, we found that inhibition of the 26S proteasome through Bortezomib treatment does not increase V194M-FLT3 protein expression. We concluded that the degradation process of mutated FLT3 seems to be independent of the functionality of the proteasomes. Furthermore, we successfully established two new induced pluripotent stem cell lines (iPSC) from the index CN patient to study early hematopoietic development. Multiple characterization experiments have shown this cell line to bear all characteristics of iPSCs. We have found FLT3 to be expressed less in the early embryoid body (EB) cells derived from patient iPSCs compared to healthy donor EBs. Late hematopoietic differentiation of index patient and healthy donor iPSCs have shown disparities in morphology vs. antigen expression profile: Both healthy donor and index patient iPSCs differentiation results in sufficient neutrophil production. However, after day 19 of EB formation, cells, while being viable, CD45 and CD11b positive, express limited CD15 and CD16. Reasons might be found in the specific cytokine stimulation and cell maintenance protocols. The monocytosis, the index patient is known for, is reflected in the surface marker analysis of the in vitro cell lines.

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