Macrophages (Mφ) are one of the most important immune cells in the body and are distributed in most tissues and organs. These cells play a central role in the non-specific immune clearance of bacteria, viruses, fungal pathogens, and specific immune response antigen presentation and corresponding cytokine production [1]. While traditionally tissue-resident, Mφ have been regarded as being continuously replenished from hematopoietic stem cells via the intermediate step of peripheral blood monocytes, this concept has recently been challenged [2].Thus, studies have recently demonstrated that substantial populations of Mφ are prenatally seeded and exhibit considerable longevity (months to years); in addition, these cells have, at least in part, self-renewal potential [3, 4].

Additionally, CD34+ hematopoietic stem cells, monocytes and early T lymphocytes, etc. [5] can be differentiated into macrophages under certain conditions. There are two main sources of human macrophages in vitro. One source is the cell lines derived from tumors, such as U937 and THP-1, and the other source is primary cells, such as peripheral blood mononuclear cells. The macrophages derived from the former have the potential for unlimited replication and play an important role in macrophage-related biology research [6, 7]. However, compared with primary macrophages, these immortalized cell lines are prone to abnormal genetic structure changes, leading to a lack of function, severely limiting their application in related research [6, 7]. Macrophages derived from peripheral blood represent macrophages that cannot self-renew, and each study requires large amounts of blood from the donor and depends on the donor’s physiological state and genes, etc., resulting in test results that are not representative.

Following the groundbreaking report by Takahashi and Yamanaka in 2006 [8], induced pluripotent stem cells (iPS) provide a new method and pathway for the screening of drugs and individual-specific treatments. Studies have demonstrated that humans, mice, pigs, sheep, guinea pigs, rhesus monkeys, and marmosets [913], and other species of various somatic cells, such as fibroblasts, peripheral blood cells, and amniotic fluid cells [14, 15], can be reprogrammed into iPS without the limitation of the donor age [16]. Now, human iPS (hiPS) can easily be generated by a number of reprogramming techniques and from various cell and tissue sources. Compared with traditional stem cell technology used in research, iPS technology not only avoids the ethical and moral limitations of stem cell research, but could also create an inexhaustible source of cells that could be used to derive the differentiated cells required for patient-specific therapy, such as macrophages, cardiomyocytes, neurons, and pancreatic beta cells [17, 18]. However, successful iPS-based gene and cell therapy relies on the efficient differentiation of iPS into the desired cell types. Whereas substantial progress has been made to differentiate hiPS into defined mature hematopoietic cells, such as granulocytes, macrophages, erythrocytes, megakaryocytes or dendritic cells, lymphoid differentiation or the generation of long-term repopulating hematopoietic stem cells from humans remain hampered by the low quality and inefficient quantity of the output cells [2].

Studies have shown that iPS can be differentiated into immune cells under appropriate induction conditions [1921]. The macrophages in immune cells are the most plastidic cells in the hematopoietic system and exist in all tissues and play many different roles in development [22], in vivo balance, tissue repair, immune response to pathogens [23, 24], and primary ovarian insufficiency [25]. Thus, the use of iPS-derived macrophages to establish a related disease model can further analyze the relationship between macrophages and disease. Although, many methods such as direct differentiation by the addition of different factors [26], the embryoid body (EB) formation method [27, 28], and co-culture with bone marrow stromal cells (OP9) [17] have been used for the differentiation of iPS into macrophages, the efficiency remains low, and the differentiation system remains unstable.

Furthermore, the generation of hiPS-derived macrophages focuses on the application of therapy or the pathogenesis of cancer [29, 30], Mendelian disease [31], Alzheimer’s disease [30], HIV [32], Chlamydia trachomatis infections [33], chronic granulomatous disease [34], and X-linked chronic granulomatous disease [35]. Unfortunately, many questions about the mechanisms of hiPS-derived macrophages in disease pathogenesis remain. Furthermore, macrophages show great promise in disease pathogenesis, particularly tuberculosis. Tuberculosis is a zoonotic infectious disease and a serious threat to human health. As the main host cells to invasive Mycobacterium tuberculosis (MTB), macrophages interact with MTB, playing a crucial role in the occurrence and development of tuberculosis. Studies of these interactions have confirmed a crucial role for these cells in the occurrence and development of tuberculosis. However, there is no information about hiPS-derived macrophages in response to tuberculosis infection. In particular, their effects on tuberculosis infection, especially the immunological function in response to tuberculosis infection, have not been thoroughly investigated.

Thus, in the present study, we optimized the method used to generate these cells by using an EB-forming method combined with the addition of different factors to differentiate iPS into monocytes and subsequently mononuclear cells into macrophages. These investigations led to development of a stable experimental culture condition for human iPS differentiation. Using Western blot analysis, immunostaining and through a combination of flow cytometric analyses, we elucidated the immunological function of hiPS-derived macrophages (iPS-Mφ) in response to Bacillus Calmette-Guérinin (BCG) a similar manner to Mφ derived from human monocyte cells.