Pluripotent stem cells including induced pluripotent stem (iPS) cells and embryonic stem
(ES) cells are known for their distinctive property of indefinite self-renewal in an
undifferentiated status, with the potential to differentiate into all types of cells. Current
protocols used in the differentiation of human iPS cells and human ES cells towards
erythropoiesis utilize two main approaches: (1) embryoid body (EB) formation, which
influences heterogeneity of the produced population, and/or (2) co-culture with mouse
stromal cells, where obstacles of purification of the cells rise, which makes the xeno-free
culture requirement difficult to achieve, in addition to the cytokine supplements.
Moreover, these protocols reported low efficiency in number and functionality,
especially with human iPS cells, and required long culture times. One of the major
challenges in erythroid cell production from human ES/iPS cells is achieving full
maturation and enucleation of erythroid cells in serum-free and feeder-free condition, in
order to ensure a completely xeno-free culture condition suitable for clinical applications.
In this study, we have designed a novel protocol for direct differentiation of human iPS
cells towards erythroid cells under serum-free conditions bypassing the EB formation
step without requiring co-culture. Our protocol involves three steps: (1)
hematopoietic/erythropoietic induction, followed by (2) erythroid differentiation, and
finally, (3) erythroid maturation and enucleation. Differentiated cells were separated into
normoxia and hypoxia conditions. As early as day 7 of culture, an early hematopoietic
marker, CD34, was observed, followed by a high expression of CD45, which is a pan
leukocyte marker in parallel to less expression of an early erythroid marker, CD71. Over
the culture period, an increase in the expression of the late erythroid marker, CD235a,
was monitored, which reached high levels by the end of the 28-day culture protocol.
Further studies on functional and morphological analysis using CFU assay showed that
the cell population on day 14 were able to form erythroid progenitor colonies, i.e. BFUEs.
Immunocytochemical staining showed the presence of heme-containing proteins,
which was later confirmed by globin expressions by qPCR. Interestingly, staining with new
methylene blue confirmed reticulocyte morphology, which indicated that partial
maturation was achieved. Hypoxia condition is a key regulator for erythropoiesis and
haemoglobin formation, as indicated by the BFU-Es formed under hypoxic conditions, together with formation of adult-type haemoglobin, as shown in qPCR. Further studies
on maturation of those cells are required in order to achieve fully mature and functional
erythroid cells phenotype. This thesis thus presents a direct differentiation protocol
toward erythroid cells using human iPS cells in serum-free and feeder-free system,
bypassing EB-stage and resulting on high efficiency of erythroid cells formation within 4
weeks of culture, which include partial maturation and formation of adult-type
haemoglobin.