Human pluripotent stem cells: an emerging model in developmental biology

Abstract

Developmental biology has long benefited from studies of classic model organisms. Recently, human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, have emerged as a new model system that offers unique advantages for developmental studies. Here, we discuss how studies of hPSCs can complement classic approaches using model organisms, and how hPSCs can be used to recapitulate aspects of human embryonic development 'in a dish'. We also summarize some of the recently developed genetic tools that greatly facilitate the interrogation of gene function during hPSC differentiation. With the development of high-throughput screening technologies, hPSCs have the potential to revolutionize gene discovery in mammalian development.

Fig. 1.

The derivation of hESCs and hiPSCs. (A) Human embryonic stem cells (hESCs) are derived from the inner cell mass of cultured preimplantation human blastocysts. When grown on mouse embryonic fibroblast feeders, human pluripotent stem cells (hPSCs) can self-renew indefinitely in culture while maintaining the ability to become derivatives of all three germ layers. (B) Human somatic cells can be reprogrammed into human induced pluripotent stem cells (hiPSCs) by: (1) ectopic expression of transcription factors; (2) ectopic expression of transcription factors together with small molecules; and (3) ectopic expression of microRNAs. These reprograming factors can be delivered into somatic cells via viral infection, transposon transgenesis, plasmid transfection and direct delivery of cell-permeable proteins or synthetic mRNAs.

Fig. 2.

Directed differentiation of hPSCs. In vitro differentiation of human pluripotent stem cells (hPSCs) can be performed in adherent culture or in suspension culture via embryoid body (EB) formation. In both formats, differentiation can be induced by treatment with growth factors and small molecules to activate or inhibit various signaling pathways in a step-wise manner by mimicking embryonic development. Typical differentiation protocols are illustrated using three specific examples: motoneurons from the ectoderm (Li et al., 2005; Wichterle et al., 2002), erythropoietic cells from the mesoderm (Niwa et al., 2011) and intestinal cells from the endoderm (Spence et al., 2011). In each case, the signaling factors and pathways required (or those that need to be inhibited) to drive differentiation into the appropriate cell types are indicated. BMP, bone morphogenetic protein; EGF, epidermal growth factor; EPO, erythropoietin; FGF, fibroblast growth factor; FP6, interleukin 6 (IL6) and IL6 receptor fusion protein; IL, interleukin; RA, retinoic acid; SCF, Kit ligand; SHH, sonic hedgehog; TPO, thyroid peroxidase; VEGF, vascular endothelial growth factor.

Fig. 3.

Advancing developmental biology and regenerative medicine through studies of hPSCs and model organisms. Genetic studies, including genetic screens and loss- and gain-of-function (LOF and GOF) studies, from the mouse and other model organisms have identified many genes and signaling pathways that govern various aspects of development. Such information has guided the search for defined conditions to turn hPSCs into specific cell types of all three germ layers (Ec, ectoderm; Me, mesoderm; En, endoderm). With the development of new genetic tools, it is now possible to use hPSCs as a new model system for studies of human development. The generation of desired cell types from hPSCs will also advance regenerative medicine in several aspects, including cell replacement therapy, disease modeling and drug discovery. shRNA, short hairpin RNA; siRNA, small-interfering RNA.

Fig. 4.

hPSC-based high-throughput screening. High throughput screening in human pluripotent stem cells (hPSCs) using chemical or RNAi libraries can be performed in arrayed (A) or pooled (B) format. (A) In arrayed screens, chemicals, small-interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) pre-arranged in multi-well plates are applied to hPSCs, and the effect can be examined by high-content imaging or by reporter assays. (B) Although pooled screens have not yet been reported using hPSCs, we envision it is possible to perform such screens using pooled shRNA viruses. Transduced cells can be further differentiated into the cell type of interest, and the target cell population can be isolated using cell surface markers or fluorescent reporters. To identify genes that inhibit or promote the lineage commitment, respectively, microarrays or next generation sequencing (NGS) can then be used to identify any over- or under-represented shRNAs (represented by red and green dots, respectively) in target cells compared with the reference cell population (e.g. the transduced cells before differentiation). FACS, fluorescence-activated cell sorting.