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Titill
en

Modulation of ER stress in HSCs

Skilað
Maí 2014
Útdráttur
en

Background: Hematopoietic stem cells (HSCs) give rise to mature blood cells and are used in bone marrow transplantation (BMT) to treat various blood malignancies. However, substantial mortality and morbidity is still related to BMT both in terms of engraftment failure and graft versus host disease. Some patients do not have the option of BMT because suitable donors can not be found or are identified too late. Ex vivo expansion of HSCs would counter against these problems but HSCs show limited growth capacity ex vivo, with drastic reduction in their reconstitution potential after a short time in culture. Knowledge in HSC regulation is vital in the development of HSC expansion from human cord blood enabling these samples to be used for effective BMT of adult patients. Recently this lab found that DPPA5 RNA binding protein plays a role in lowering ER stress and increasing HSC capacity after ex vivo culture. Sensing of ER stress relies on posttranslational changes in low abundant proteins and studying this in detail would not be possible in primary HSCs so we asked if we could use the LHX2 HSC-like cell line as a model for ER stress in the hematopoietic system.
Methods: ER stress was induced in LHX2 cells with tunicamycin (TM). They were then analyzed with fluorescence activated cell sorter (FACS) and ER stress proteins were analyzed with Western Blot. Lentiviral knockdown (KD) of DPPA5 was performed in the cells and RNA was analyzed with qRT-PCR.
Results: LHX2 cells induced with TM showed dose dependant lowering in both total cell number and in multipotent progenitor cell numbers. Cells that were treated with the highest dose of TM had an increase in CHOP (late time point ER stress inducer) which promotes cell apoptosis. DPPA5 KD cells showed marked decrease in LSK cell number. The DDIT3 gene, that codes for the CHOP protein, was upregulated in these cells suggesting that they were more sensitive to ER stress.
Disscusion: The LHX2 cell line is very sensitive to induced ER stress. The LHX2 cells express DPPA5 and they may be used to look at specific targets of DPPA5. We showed that the DPPA5 KD cells have increased expression of genes related to ER stress but to see the full extent of the effects we would need to further analyse the effects of the DPPA5 KD and effects on ER stress.

Samþykkt
13.5.2014


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