Application Focus: New Methods for iPSC Differentiation, Inducing a Mammary Fate

Discovery of the Key to Pluripotency

Induced pluripotent stem cells (iPSCs) may be generated from a wide range of fully differentiated cells, and under optimal conditions may be prompted to differentiate into virtually any fate. Induced stem-like cells not only provide an alternative to embryonic stem cells, but more importantly represent powerful tools for drug development and disease modeling.¹

Methods for the induction of pluripotency were developed in 2006, when genes critical for cellular reprograming were identified by Yamanaka and Takahashi, including OCT4, SOX2, c-MYC, and KLF4.² Expression of these four factors proved sufficient to induce iPSCs from human fibroblasts.³ In combination, these genes control cell proliferation-, suppress differentiation- and regulate epigenetic-programs, leading to a pluripotent state. Since the discovery of these key factors, the exact combination of genes and methods used to induce pluripotency have continued to evolve with the goal in sight of improving both efficiency and safety for eventual applications in the human.^1,4

Methods for the Induction of Pluripotent Cells

Traditionally, iPSCs were first generated by expression of genes transduced into somatic cells via viral vectors.^1,2,3 However, for ultimate application in patients, this method presents the caveat of foreign genetic material becoming integrated into the genome. Therefore, new approaches have focused on the use of plasmid DNA and mRNA for the expression of genes controlling pluripotency. Additionally, protein-based reprogramming approaches circumvent the introduction of foreign genetic material. Recombinant proteins may be generated and purified using bacterial systems or mammalian cells.5 However, proteins must be modified to gain access to the cell and the nucleus. Therefore, inclusion of specific sequences including TAT protein, derived from the human immunodeficiency virus transactivator of transcription (HIV-TAT), allows pluripotency inducing factors (OCT4, SOX2, c-MYC, and KLF4) to reach the nucleus and target key promoter regions.⁵ Poly-arginine fused pluripotency inducing factors have also been effective in gaining access into cells and inducing iPSCs.⁵ Overall, the discovery of these four key transcription factors continues to propel the development of new strategies focused on the use of iPSCs in regenerative and personalized medicine.

FGF acidic was detected in immersion fixed paraffin-embedded sections of human breast using Goat Anti-Human FGF acidic Antigen Affinity-purified Polyclonal Antibody (Catalog # AF232) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to epithelial cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Modelling Mammary Tissue from iPSCs

Recently, a new method for the differentiation of human iPSCs into mammary organoids has been developed using a combination of factors essential for embryonic mammary gland development.⁶ For this new two-step approach, researchers first induced hiPSCs to predominantly adopt a non-neuronal ectoderm patterning.  Using conditioned media for mammary differentiation, investigators developed suspension sphere cultures to generate embryoid bodies (mEBs) enriched for the expression of non-neuronal ectoderm markers including CK8, CK18, AP-2alpha, AP-2gamma and P63. Microarray analysis of mEBs cultured for 10 days demonstrated the expression of molecular markers which support mammary differentiation programs.

In the second step, investigators used mEBs and a 3D culture system, which consisted of a combination of Matrigel (see Cultrex for a superior alternative) and Collagen I, to generate mammary-like organoids. Cultures were supplemented with various factors, as shown in table below, to promote mammary development and function. Under these conditions, mEBs developed alveolar mammary morphology starting at 10 days, which became more complex by 30 days.

Finally, investigators confirmed the expression of several breast markers including a-lactalbumin/LALBA, EpCAM, CK14 and P63, demonstrating that organoids grown under the specified conditions differentiated a mammary fate.

This study represents the first report of methods for the differentiation of hiPSCs towards the formation of mammary organoids. Although these methods failed to replicate specific mammary morphologies of interest (e.g., two-layer mammary ducts), the identification of factors required to direct mammary differentiation from hiPSCs is sure to further efforts for the modelling and treatment of breast disease.

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References

1. Orqueda, A. J., Giménez, C. A., & Pereyra-Bonnet, F. (2016). IPSCs: A Minireview from bench to bed, including organoids and the crispr system. Stem Cells International. Hindawi Publishing Corporation. https://doi.org/10.1155/2016/5934782
2. Takahashi, K., & Yamanaka, S. (2006). Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell, 126(4), 663–676. https://doi.org/10.1016/j.cell.2006.07.024
   
3. Takahashi, K., Okita, K., Nakagawa, M., & Yamanaka, S. (2007). Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc, 2(12), 3081–3089. https://doi.org/10.1038/nprot.2007.418
   
4. Yoshida, Y., & Yamanaka, S. (2010, July 6). Recent stem cell advances: Induced pluripotent stem cells for disease modeling and stem cell-based regeneration. Circulation. https://doi.org/10.1161/CIRCULATIONAHA.109.8814335. doi: 10.1387/ijdb.140007XL
5. Li, X., Zhang, P., Wei, C., & Zhang, Y. (2014). Generation of pluripotent stem cells via protein transduction. The International Journal of Developmental Biology, 58(1), 21–7. https://doi.org/10.1387/ijdb.140007XL
6. Qu, Y., Han, B., Gao, B., Bose, S., Gong, Y., Wawrowsky, K., … Cui, X. (2017). Differentiation of Human Induced Pluripotent Stem Cells to Mammary-like Organoids. Stem Cell Reports, 8(2), 205–215. https://doi.org/10.1016/j.stemcr.2016.12.023