Megacult

CD34+ HSPCs were lentivirally transduced on RetroNectin-coated (Takara) plates as described [32 (link)]. Methylcellulose-based (Methylcellulose Base and Complete, RnD Systems) and collagen-based (Megacult®, Stem Cell Technologies) colony-forming assays were carried out according to the manufacturers’ instructions. Serial replating was performed as described previously [33 (link)]. Cumulative colony numbers were calculated with the following equation: $\mathrm{CFU}\left(\mathrm{k}\right)={\displaystyle \sum _{\mathrm{n}=1}^{\mathrm{k}}{\mathrm{CFU}}_{\mathrm{n}}}$ , where CFU_n = number of counted colonies from respective platings (n). Note that if a fraction of cells (f) from the 1^st plating was replated for the 2^nd plating, then $\mathrm{CFU}\left(\mathrm{k}\right)={\mathrm{CFU}}_1+\frac{{\mathrm{CFU}}_2}{{\mathrm{f}}_1}$ .

FACS-purified cells were sorted into FBS, spun down at 300 g, and then plated into either methylcellulose, BFU-E, or MegaCult media as per the manufacturer’s protocol. For methylcellulose colony assays, FACS-purified c-kit⁺ (5,000 per well) bone marrow cells were plated in HSC007 methylcellulose (R&D Systems). For BFU-E assays, FACS-purified MEP (3,500 per well) or c-kit⁺ cells treated with vehicle or Heparinase I, II, III (3,500 per well) were sorted and plated in HSC006 methylcellulose (R&D Systems) supplemented with 50 ng/ml SCF, 20 ng/ml IL-3, 20 ng/ml IL-6, and 10 U rhEPO. Colonies were then counted 7 d later. For MegaCult assays, FACS-purified c-kit⁺ (3,500 per well) or MEP (3,500) mouse bone marrow cells were sorted and plated in MegaCult media as per manufacturer protocol (STEMCELL Technologies). Human CD34⁺ cells from mobilized peripheral blood were allowed to recover in StemSpan II media with cytokines (SCF, IL-3, Flt,IL-6, and TPO all at 50 μg/ml) for 12 h before sorting, and human MEPs (3,000–4,500 per condition) were sorted and plated in MegaCult. For mouse samples, colonies with three or more megakaryocytes were scored 8 d later based on acetylcholinesterase positivity following manufacturer’s protocol. For human samples, slides were stained with the MegaCult Human Staining Kit as per manufacturer protocol.

Sorted CRISPR-modified HSPCs were seeded either in methylcellulose media (MethoCult™ Enriched, triplicates, 300 cells each) or collagen-based media w/or w/o 50 ng/ml TPO (MegaCult™, quadruplicates, 1800 cells each, both StemCell Technologies) and incubated for 12–14 days. Collagen embedded colonies were fixed and stained magenta using a mouse anti-CD41 primary antibody (Biolegend, San Diego, CA, USA) followed by an anti-mouse alkaline phosphatase (AP) secondary antibody and AP substrate (Vector Laboratories, Burlingame, CA, USA). Colonies were counted and scored based on morphology and staining.