Induced pluripotent stem cells (iPSCs) provide an ideal starting point for the generation of gene-edited tissue-specific cells. In particular, engineered iPSC-derived mesenchymal stromal cells (iMSCs) combine the advantages of tissue-derived MSCs (immunomodulation, clinical safety, low immunogenicity, tissue penetration, etc.) with gene editing (e.g., overexpression or knock-out of immunomodulatory factors), and thus, presents anew pathway for the development of cell therapies with higher efficacy and defined modes of action. iMSCs readily overcome the clonality, viability and expandability challenges associated with tissue-derived MSCs and display greater immunomodulation even in their naive form. Engineered iMSCs (EiMSCs) that overexpress transgenic immunomodulatory cytokines could offer further therapeutic potential. Although the overexpression of transgenic cytokines in EiMSCs presents an exciting therapeutic opportunity, transgenic expression can be unstable or silenced during the differentiation of iPSCs to EiMSCs. To enable sustained transgene expression upon EiMSC differentiation, we explored the use of universal chromatin-opening elements (UCOEs) in transgenic cassettes that express green fluorescent protein (GFP) under the EF1α promoter. Cassettes with and without a UCOE were inserted into the adeno-associated virus integration site 1 (AAVS1) safe-harbor locus in iPSCs through the use of mRNA encoding UltraSlice™ gene-editing endonucleases and single-stranded DNA (ssDNA) repair templates. Clonal iPSC lines were isolated by the single-cell deposition of GFP-sorted cells, biallelic insertion into AAVS1 was verified by amplicon sequencing, and GFP expression during differentiation toward EiMSCs was monitored. EiMSC lines all displayed traditional MSC surface markers upon differentiation, but they showed differences in GFP expression: While only ~30% of EiMSCs engineered without a UCOE 6-3 were GFP-positive, use of a UCOE yielded in uniform GFP expression (>99%) that persisted after 10 passages and 2 freeze-thaw cycles. Inflammatory (e.g., IL7 and IL15) and anti-inflammatory cytokines (e.g., IL4 and IL10) are being explored for their opposing, but potentially crucial roles in MSC therapy by directing and enhancing immunomodulation and survival of immune cells to alleviate conditions such as cancer and inflammatory disease, respectively. We therefore applied our established workflow to knock-in expression cassettes encoding either IL7-IL15 or IL4-IL10 fusion proteins into iPSCs and differentiate them to clonal EiMSC lines. Taking advantage of the high efficiencies obtained for UltraSlice™-mediated knock-ins, clonal iPSC lines were successfully established via single-cell deposition without the use of a reporter system. MSC markers and transgene expression were confirmed in each line upon differentiation, yielding two EiMSC lines that stably express IL7-IL15or IL4-IL10 fusion proteins. These lines may prove promising in the development of therapies to treat a wide range of diseases.