RH-1

The Differentiation Protocol Allows the In-Vitro Generation of Erythroid Cells from a Wide Variety of Human Stem Cells

Plasmodium falciparum's clinically relevant blood stage involves erythrocyte invasion, yet studying host genetic factors is limited by erythrocytes' anuclear nature. Pance et al. overcame this by generating erythroid cells from stem cells using an in vitro differentiation protocol, combining flow cytometry-based hemozoin detection with patient-derived iPSC reprogramming and genome editing.

The objective was establishing a protocol to produce sufficient erythroid cells for invasion assays. To minimize cell loss, embryoid body stages and co-cultures were avoided, instead directing cells toward mesoderm through two cytokine steps based on Vallier et al. (2009). First, low Activin A with FGF2 inhibition suppressed neuroectoderm; then BMP4, FGF2, and SB431542 (Activin A inhibition) favored mesoderm over endoderm. IL-3 was added to direct mesoderm toward haematopoiesis. qRT-PCR showed declining pluripotency markers and rising mesoderm markers (Fig. 1A), followed by induction of megakaryocyte-erythroid progenitor transcription factors, coinciding with morphological changes.

Microarray analysis confirmed transcriptome transition from early mesoderm to haematopoiesis (Fig. 1B). Mesoderm specification genes (eomes, BMP2/4, BMPR1B, CFC1) peaked during the Meso-Ery transition, while haematopoietic drivers (T, Tal1, GATA1, MIXL1) peaked later. Based on haematopoietic gene expression, this stage was set at 8-12 days, with cells spontaneously detaching for harvest without trypsinisation.