The generation of human macrophages from induced pluripotent stem cells (iMacs) is a rapidly developing approach used to create disease models, screen drugs, study macrophage–pathogen interactions and develop macrophage-based cell therapy. To generate iMacs, different types of protocols have been suggested, all thought to result in the generation of similar iMac populations. However, direct comparison of iMacs generated using different protocols has not been performed. We have compared the productivity, the differentiation trajectories and the characteristics of iMacs generated using two widely used protocols: one based on the formation of embryoid bodies and the induction of myeloid differentiation by only two cytokines, interleukin-3 and macrophage colony-stimulating factor, and the other utilizing multiple exogenous factors for iMac generation. We report inter-protocol differences in the following: (i) protocol productivity; (ii) dynamic changes in the expression of genes related to inflammation and lipid homeostasis following iMac differentiation and (iii) the transcriptomic profiles of terminally differentiated iMacs, including the expression of genes involved in inflammatory response, antigen presentation and lipid homeostasis. The results document the dependence of fine iMac characteristics on the type of differentiation protocol, which is important for further development of the field, including the development of iMac-based cell therapy.

Induced pluripotent stem cells (iPSCs) can be generated from various adult cells, genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however, their systemic administration can lead to severe adverse outcomes. One way of overcoming the limitation is to introduce cells able to enter the site of pathology and to produce IFN-Is locally. As a first step towards the generation of such cells, here, we aimed to generate human iPSCs overexpressing interferon-beta (IFNB, IFNB-iPSCs). IFNB-iPSCs were obtained by CRISPR/Cas9 editing of the previously generated iPSC line K7-4Lf. IFNB-iPSCs overexpressed IFNB RNA and produced a functionally active IFN-β. The cells displayed typical iPSC morphology and expressed pluripotency markers. Following spontaneous differentiation, IFNB-iPSCs formed embryoid bodies and upregulated endoderm, mesoderm, and some ectoderm markers. However, an upregulation of key neuroectoderm markers, PAX6 and LHX2, was compromised. A negative effect of IFN-β on iPSC neuroectoderm differentiation was confirmed in parental iPSCs differentiated in the presence of a recombinant IFN-β. The study describes new IFN-β-producing iPSC lines suitable for the generation of various types of IFN-β-producing cells for future experimental and clinical applications, and it unravels an inhibitory effect of IFN-β on stem cell neuroectoderm differentiation.

Maturity-onset diabetes of the young type 10 (MODY10) is inherited in an autosomal dominant manner, characterized by islet cell dysfunction, impaired insulin synthesis and secretion. MODY10 is relatively rare, and is caused by a mutation of the 11p15.5 site on chromosome 11 encoding insulin. We obtained iPSCs (ERCi003-A line) from dermal fibroblasts derived from a patient with MODY10 carrying a previously undescribed pathogenic heterozygous mutation in the INS gene (c.93C>G, p.C31W) into iPSCs using transfection with self-replicating RNA vector. iPSCs proliferate in dense monolayer cell colonies, have a normal karyotype (46,XY), express pluripotency markers (OCT4, SOX2, TRA-1-60). The functional pluripotency of iPSCs was confirmed by their ability to form embryoid bodies and differentiate into the three germ layers (ecto-, endo-, and mesoderm). Sanger sequencing of iPSCs confirmed the presence of pathogenic heterozygous mutation in the INS gene. This cell line could be useful to thoroughly modeling of MODY10 to improve clinical understanding of the disease.

Maturity-onset diabetes of the young type 3 (MODY3) disorder is characterized by an autosomal dominant type of inheritance and highly heterogeneous clinical phenotype influenced by type and position of mutation in the HNF1A gene. We reprogrammed dermal fibroblasts derived from a patient with MODY3 carrying a heterozygous mutation in the site encoding the transactivation domain of the HNF1A protein (c. 864delGinsCC, p.Gly292ArgfsTer25) into iPSCs using transfection with self-replicating RNA vector. Obtained iPSCs (ERCi004-A line) proliferate in dense monolayer cell colonies, have a normal karyotype (46,XX), express pluripotency markers (OCT4, SOX2, TRA-1-60). The functional pluripotency of iPSCs was confirmed by their ability to form embryoid bodies and differentiate into the three germ layers (ecto-, endo-, and mesoderm). Sanger sequencing of iPSCs confirmed the presence of pathogenic heterozygous mutation in the HNF1A gene. This cell line could be useful to modeling of MODY3 pathology to improve understanding of the mechanism of the transactivation domain mutation, as well as a potential source for autologous cell-based therapy.

Allogeneic chimeric antigen receptor (CAR)-based cell products and induced pluripotent stem cell (iPSC)-derived regenerative grafts increasingly use multilayer immune engineering. Current strategies are strongest against direct human leukocyte antigen (HLA)-dependent recognition but incompletely address natural killer (NK)-cell counter-pressure, priming by recipient antigen-presenting cells, humoral and complement-mediated injury, inflammatory remodeling, blood- and tissue-interface damage, and terminal cell killing. Poxvirus research has characterized diverse immunomodulatory mechanisms relevant to these barriers. We review poxviral control of interferon signaling, antigen processing and presentation, T-cell activation, NK-cell surveillance, cytokine and chemokine networks, complement, inflammatory signaling, and regulated cell death, integrating viral mechanisms with human perturbation, cell-engineering, and transplantation evidence. A qualitative Tier 1–3 framework distinguishes mechanisms close to current cell-therapy practice from those requiring greater product-specific validation or control. The resulting design rules favor graded antigen-presentation control with paired NK validation, local or transient inflammatory buffering, interface-restricted complement protection, and survival enhancement only when independent eliminability is preserved. Selected poxviral proteins can act autonomously outside productive infection, but direct viral-protein expression remains a selective rather than default option. We propose a conditional defense-in-depth framework in which modules are added only when a residual barrier is demonstrated in the final therapeutic product.