Methocult classic

One thousand and five hundred CD34⁺ cells treated or not with KG-1 and ME-1 derived EVs, were cultured in two 35 mm dishes in MethoCult Classic (Stem Cell Technologies, Vancouver, BC, Canada) according to the manufacturer’s instruction. All type of colony forming unit (CFU), specifically Burst-Forming-Unit Erythrocyte (BFU-E), CFU Granulocyte, Erythrocyte, Macrophage, Megakaryocyte (CFU-GEMM), CFU Granulocyte, Macrophage (CFU-GM) and CFU Macrophage (CFU-M), were visualized and counted with Axio microscope (Carl Zeiss Inc., Thornwood, New York) after being cultured in incubator at 37°C and 5% CO₂ for 14 days.

FACS-sorted iPS-derived CD34 + cells from the CMS and non-CMS subjects were plated at a density of 10⁵ cells per 35 mm dish combined with MethoCult Optimum media (Stemcell Technology, Cat. H4034) and 2% FBS (Sigma, Cat. F2442). Dishes were incubated at 37 °C in an incubator with 5% CO₂ and 5% O₂ for 14 d, at which time colonies were scored for BFU-E and CFU–GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte). For EPO dose-response experiments, Methocult classic (without EPO) (Stemcell Technology, Cat. H4434) was mixed with various doses of EPO (0.5, 1, 1.5, 2, 2.5, 3 and 3.5 units) and assayed for colony production. We used N = 4 subjects for each group that was tested by this assay.

CD34⁺ cells which had undergone DNAm targeting alone or dual genetic and epigenetic editing were cultured in StemSpan II supplemented with cytokines for 24 h before seeding into the CFU assay. CD34⁺ cells were counted, and 600 cells were seeded per plate into MethoCult Classic (StemCell Technologies) after DNAm editing, or 1,200 cells per plate after dual editing. Colonies were grown in an incubator for 14 d before counting and harvesting either bulk or single colonies. For single-colony harvest, individual colonies were picked using an EVOS light microscope (ThermoFisher Scientific) and deposited directly into a PCR tube for analysis.

HSPC potential to proliferate as colonies and differentiate into myeloid lineages was evaluated before and after HSPC expansion through the CFU assay. 1000 non-expanded CD34⁺-enriched cells (day 0) or 2500 expanded cells (day 7) were resuspended in 2 mL of MethoCult™ Classic (STEMCELL Technologies) medium, divided into three wells of a 24-well culture plate and left for 14 days to incubate at 37 °C, 5% CO₂ in a humidified atmosphere [27 (link)]. After an incubation period of 14 days, multilineage colony-forming unit (CFU-Mix), burst-forming unit erythroid (BFU-E), and colony-forming unit granulocyte-macrophage (CFU-GM) colonies were classified and counted using a brightfield microscope (Olympus CK40 (Olympus, Tokyo, Japan)). Colony number was divided by the number of seeded cells and then multiplied by the number of expanded or non-expanded HSPC. Fold change in total colony number (FC Total CFU) was obtained by dividing the total colony number at day 7 by the respective of day 0.

HMCLs (2 × 10³) either treated or untreated with PTC-209 at 1 μM were plated in duplicates in 1.1 ml methylcellulose-based medium (MethoCult Classic, StemCell Technologies) per 6-well and incubated for 14 days (37 °C, 5 % CO₂). At the end of the incubation period, the number of colonies consisting of >40 cells was scored using an inverted microscope with ×4, ×10 and ×20 planar objectives.