<em>In Vitro</em> Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Abstract

SHORT ABSTRACT We present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for efficient differentiation of human pluripotent stem cells, and can generate large quantities of cells for genetic manipulation. LONG ABSTRACT The placenta is the first organ to develop during embryogenesis, and is required for survival of the developing embryo. The placenta is comprised of various trophoblast cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions with isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development, and can also be differentiated in vitro to trophoblast cells that express markers of the various trophblast cell types. Here we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss of function phenotypes, or infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated to trophoblast progenitors for gain of function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications including drug discovery and stem cell research.

Sex-Specific Gene Expression Differences Are Evident in Human Embryonic Stem Cells and During In Vitro Differentiation of Human Placental Progenitor Cells

“…Complete differentiation of hESCs into trophoblast-like cells (EVTs, SYNs) requires 9-14 days [9]; here, we limited differentiation to days 2-5 to investigate sex-specific differences with the early commitment for differentiation into trophoblastic cells. We observed morphological changes after 1 day of BMP4/A/P differentiation, with cells appearing flatter and larger with enlarged nuclei, as previously observed [6,51]. Cellular differentiation was confirmed using quantitative PCR for the pluripotency marker NANOG, which decreased by day 2, and expression of trophectoderm markers KRT7 and CDX2, whose expression peaked at days 2-4 (data not shown), as described previously [9,51].…”

“…We observed morphological changes after 1 day of BMP4/A/P differentiation, with cells appearing flatter and larger with enlarged nuclei, as previously observed [6,51]. Cellular differentiation was confirmed using quantitative PCR for the pluripotency marker NANOG, which decreased by day 2, and expression of trophectoderm markers KRT7 and CDX2, whose expression peaked at days 2-4 (data not shown), as described previously [9,51]. We verified that CDX2 and KRT7 proteins were expressed during BMP4/A/P differentiation using IF, and these markers appeared between days 2 and 3 of differentiation (Fig.…”

Orchestrating stem cell fate: Novel tools for regenerative medicine

“…Within this context, some authors found that medium conditioned by factors such as activin A, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF) or Dickkopf-related protein 1, can optimize cardiac development in mouse and human stem cell lines[46,47]. BMP4 itself, in combination with inhibitors of the Activin/Nodal signaling pathways, induces differentiation of ESCs into trophoblastic cells, which show similar trophectoderm profile and are able to secrete placental hormones[48]. Concerning the use of chemistry to push stem cells to specific phenotypes, molecules that can affect the epigenetic code to activate a molecular differentiation program have largely been used.…”

Blastocyst-like embryo surrogates from stem cells in implantation models

“…With the use of BMP4 and the 2 inhibitors A83-01, PD173074 (BAP), the derived trophoblast-like cells express trophoblastic markers ( KRT7 , HLA-G , CGB ), secrete hormones (β-hCG, estrogen, and progesterone) and possess invasive ability [82,85] . The BAP-induced trophoblast from hESCs was reported to be a suitable model system for studying early human placentation [86] .…”