Differentiation and Characterization of CD34+ Cells

CD34+ cells from healthy donors were obtained from Lonza (Basel, Switzerland). The cells were cultured in erythroid or granulomonocytic differentiation media for 14 day as described previously.19 (link), 20 (link) For cell growth assays, on day 7 of culture transduced cells were seeded in 96 well plates (10000 cells/200µl). Viable cells were counted by using trypan blue exclusion assay. For colony assays, on day 7 of culture 3000 transduced cells were plated on methylcellulose (MethoCult H4434 Classic, Stemcell Technologies) containing 0.65µg/ml puromycin according to the manufacturer’s protocol. Colonies were counted after 14 days. May-Grünwald and Giemsa stains were used to stain cytospin slides of granulomonocytic and erythroid cells according to the manufacturer’s protocol (Sigma Aldrich).

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Chase A., Pellagatti A., Singh S., Score J., Tapper W.J., Lin F., Hoade Y., Bryant C., Trim N., Yip B.H., Zoi K., Rasi C., Forsberg L.A., Dumanski J.P., Boultwood J, & Cross N.C. (2018). PRR14L mutations are associated with chromosome 22 acquired uniparental disomy, age related clonal hematopoiesis and myeloid neoplasia. Leukemia.

Publication 2018

MethoCult H4434 Classic May-Grünwald and Giemsa stains

Assays Cells Cells culture Donors Erythroid cells Giemsa stains Methylcellulose Puromycin Stain Stemcell Trypan blue

Corresponding Organization :

Other organizations : University of Southampton, Oxford BioMedica (United Kingdom), University of Oxford, Salisbury NHS Foundation Trust, Wessex Regional Genetics Laboratory, Salisbury District Hospital, Academy of Athens, Science for Life Laboratory, Uppsala University

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Variable analysis

independent variables

Differentiation medium (erythroid or granulomonocytic)

dependent variables

Cell growth (viable cell count)
Colony formation (colony count)

control variables

CD34+ cells from healthy donors
Culture duration (14 days)
Puromycin concentration (0.65 µg/ml) for colony assay

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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