“9.61x6.69” b2815 Tissue Engineering and Nanotheranostics
Directed Differentiation of Human Pluripotent Stem Cells 93
Of the cytokines tested, BMP4 was the only one that promoted devel-
opment of a primitive streak population, one of the first differentia-
tion steps towards a hematopoietic mesoderm, and BMP4 also
promoted expression of the generation of hematopoietic progenitors
at low efficiency.^64 Addition of VEGF to the culture significantly
improved the yield of hematopoietic progenitors, and SCF and FGF2
addition further increased efficiency.^64
In 2011, Woods et al. outlined a protocol with a completely
defined media for hematopoietic induction.^65 They used this media,
containing fetal bovine serum, BMP4, TGF-β, VEGF, TPO, EPO,
SCF, Flt-3 ligand, holotransferrin, PGE2, and ascorbic acid, to cul-
ture human embryoid bodies created from embryonic and iPSCs.^65
While the initial publication required a second step where embryoid
bodies were cocultured with OP9 stromal cells, recent advances have
eliminated the need for a coculture.65,66 The commercially available
media, MesoTotal, provided efficacy in producing CD34/CD45
positive and CD38 negative hematopoietic cells in a two-step
embryoid body system without coculture.^66 Additionally, Ronn et al.
showed that inhibition of retinoic acid signaling enhanced hemat-
opoietic lineage commitment.^66
In 2016, Kitajima et al. published a successful 2D culture tech-
nique to generate hematopoietic progenitors without a coculture.^67
Their protocol initiated the differentiation by activating the Wnt/
β-catenin pathway via GSK3 inhibitor and was followed by a series of
cytokine additions to the culture.^67 They found that GSK3 inhibition
improved the efficiency of deriving cells with CD34 and CD45
markers.^67 Further refinements are still needed to clarify the essential
factors for in vitro human hematopoietic differentiation. This is
further complicated by current difficulties characterizing the true
hematopoietic stem cell state.
3.3.3 Endothelial cells
Endothelial cells derived from the mesoderm are found in the heart,
lining blood, and lymph vessels. These cells are involved in fluid filtra-
tion, recruitment of specialized blood cells, hormone trafficking, and
