Enhancing mammary differentiation by overcoming lineage-specific epigenetic modification and signature gene expression of fibroblast-derived iPSCs

Recent studies have shown that induced pluripotent stem cells (iPSCs) retain a memory of their origin and exhibit biased differentiation potential. This finding reveals a severe limitation in the application of iPSCs to cell-based therapy because it means that certain cell types are not available for reprogramming for patients. Here we show that the iPSC differentiation process is accompanied by profound gene expression and epigenetic modifications that reflect cells'' origins. Under typical conditions for mammary differentiation, iPSCs reprogrammed from tail-tip fibroblasts (TF-iPSCs) activated a fibroblast-specific signature that was not compatible with mammary differentiation. Strikingly, under optimized conditions, including coculture with iPSCs derived from the mammary epithelium or in the presence of pregnancy hormones, the fibroblast-specific signature of TF-iPSCs obtained during differentiation was erased and cells displayed a mammary-specific signature with a markedly enhanced ability for mammary differentiation. These findings provide new insights into the precise control of differentiation conditions that may have applications in personalized cell-based therapy.The mammary gland is a primary target for carcinogenesis. Breast cancer occurs at a high rate and affects one in eight women in Western countries during their lifetime.^{1, 2} In the United States alone, 232 340 new invasive breast cancer cases were reported for women in 2013 and 39 620 patients died.³ Regenerative therapy of the damaged mammary gland tissues is the best way to restore breast functions; therefore, the creation of stem cells that are capable of developing into fully functional mammary glands is desirable. There are two distinct types of pluripotent stem cells that may be used for this purpose. The first is embryonic stem cells (ESCs) derived from the inner cell mass of embryonic blastocysts,⁴ and the second is induced pluripotent stem cells (iPSCs) obtained by reprogramming somatic cells.⁵ Although, in theory, both ESCs and iPSCs can be differentiated into any type of mature cell, use of the latter is more desirable because it does not require the killing of embryos, and the cells can be derived from virtually any type of tissue. In addition, because iPSCs can be generated from the same patient, the use of iPSCs avoids the immunosuppressive reactions that have long hampered organ and tissue transplantation.^{6, 7, 8} However, recent studies have shown that some iPSCs seem to retain a memory of their origin and exhibit skewed potential during differentiation for tissue/organ formation.^{9, 10, 11, 12, 13, 14} This feature may represent a limitation if certain cell types from diseased tissues or organs are not available for reprogramming.Numerous studies about the use of ESCs have indicated that, although these cells have the potential to generate all cell types, their differentiation depends critically on many factors.^{14, 15, 16} Precise conditions are required for driving cells into specific pathways leading to new lineage formation (reviewed in Murry and Keller¹⁷ and Cahan and Daley¹⁸). Based on these observations, we hypothesized that the skewed differentiation of iPSCs could be overcome by providing favorable conditions for differentiation. To test this hypothesis, we have generated iPSCs from mouse mammary epithelial cells (ME-iPSCs) and mouse-tail fibroblasts (TF-iPSCs), and have studied the gene expression profiles and epigenetic modifications during differentiation. We found that, although these iPSCs activate distinct signature memories that are reflective of their origins during the differentiation process, the fate of iPSCs could be redirected under optimized conditions in favor of the formation of a desired tissue/organ.