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.

Macrophages play a crucial role in the development and control of inflammation. Understanding the mechanisms balancing macrophage inflammatory activity is important to develop new strategies for treating inflammation-related diseases. TNF-α-induced protein 3 (TNFAIP3, A20) is a negative regulator of intracellular inflammatory cascades; its deficiency induces hyper-inflammatory reactions. Whether A20 overexpression can dampen macrophage inflammatory response remains unclear. Here, we generated human-induced pluripotent stem cells with tetracycline-inducible A20 expression and differentiated them into macrophages (A20-iMacs). A20-iMacs displayed morphology, phenotype, and phagocytic activity typical of macrophages, and they displayed upregulated A20 expression in response to doxycycline. A20 overexpression dampened the A20-iMac response to TNF-α, as shown by a decreased expression of IL1B and IL6 mRNA. A dynamic analysis of A20 expression following the generation of A20-iMacs and control iMacs showed that the expression declined in iMacs and that iMacs expressed a lower molecular weight form of the A20 protein (~70 kDa) compared with less differentiated cells (~90 kDa). A low-level expression of A20 and the predominance of a low-molecular-weight A20 form were also characteristic of monocyte-derived macrophages. The study for the first time developed a model for generating macrophages with an inducible expression of a target gene and identified the peculiarities of A20 expression in macrophages that likely underlie macrophage preparedness for inflammatory reactivity. It also suggested the possibility of mitigating inflammatory macrophage responses via A20 overexpression.

A variety of cells can be applied as vectors for the targeted delivery of chemotherapeutic or gene therapeutic agents to neoplasms. Macrophages are regarded as promising candidates for cell-based therapy. Accurate assessments of the efficacy and safety profiles of cell-based therapy products require the collection of data on their biodistribution and fate. The study of living cell distribution in vivo necessitates the utilization of a combination of methodologies to obtain more precise data regarding the fate of cells after their administration into animals. In the present study, a murine RAW 264.7 cell line was engineered to express enhanced green fluorescent protein (GFP). These cells were labeled with 50 nm magnetic nanoparticles (MNPs) for non-invasive real-time monitoring in mice using the magnetic particle quantification (MPQ) technique. The combination of high sensitivity and multimodality of the approach used permitted the acquisition of comprehensive data on the biodistribution of RAW-GFP cells in mice. For the first time, non-invasive, real-time monitoring of the dynamics of MNP-loaded macrophages in the bloodstream of mice has been achieved via the MPQ technique. Following intravenous administration, the cells are rapidly eliminated from the bloodstream, with subsequent accumulation mainly in the lungs and the liver. This may impose limitations on the use of such cells for drug delivery to other regions of a living organism.

Hematopoietic stem cells (HSCs) have long been one of the most well-studied type of stem cells. Multiple experimental and clinical studies allowed for characterizing the main HSC properties and elaborating methodological approaches for their analyses and ultimately led to the formulation of a hierarchical theory of hematopoiesis. According to the hierarchical theory, HSCs stand at the root of the hematopoietic tree and are characterized by high self-renewal and multipotent differentiation capacities. Following the differentiation, HSC successively give rise to diverse hematopoietic precursors that ultimately produce the whole variety of differentiated blood cells. The concept on the strict sequence of HSC differentiation has recently changed, largely due to the development and implementation of new hi-tech methods of cell differentiation analysis. This review summarizes classical views on HSC biology and early stages of their differentiation, outlines the main methodological approaches used in HSC studies, and discusses recent achievements and modern views on the early stages of hematopoiesis.

Fetal liver stroma consists of different cell populations. We found that the liver of 17- and 20-day rat fetuses contained skeletal muscle precursors that expressed MyoD. In primary cultures of liver cells from 15-, 17- and 20-day fetuses, spontaneous myotube formation was observed. The antigenic profile of these myogenic elements assayed by immunocytochemistry and PCR unambiguously indicated their skeletal muscle nature. Examination of major myogenic gene expression demonstrated that myogenic potencies cells from liver depended on the stage of fetal development cell cultivation. It was shown that fetal liver MSCs were capable of myotube formation in induction medium with 5-azacytidine. The results of our study show that 15- to 20-day prenatal rat liver contains mainly preexisting skeletal muscle precursors expressing MyoD and, probably, inducible muscle precursors.

FIBROBLASTY KOZhI ZARODYShEY I IKh UChASTIE V REGENERATsII KOZhNYKh RAN I SKELETNYKh MYShTs ФИБРОБЛАСТЫ КОЖИ ЗАРОДЫШЕЙ И ИХ УЧАСТИЕ В РЕГЕНЕРАЦИИ КОЖНЫХ РАН И СКЕЛЕТНЫХ МЫШЦ