Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function.

Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies.

Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors.

Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into 'induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors.

Dissecting hematopoietic differentiation using the embryonic stem cell differentiation model.

Embryonic stem cells (ESCs) have been successfully used to study the generation of the hematopoietic lineage. The ESC differentiation model provides access to distinct developmental stages during hematopoietic differentiation enabling us to study developmental transitions in a manner that is difficult to do with embryos. The identification of the bipotential hemangioblast/blast-colony forming cell (BL-CFC) which represents the earliest stage of hematopoietic commitment in ESC cultures has enabled the study of signalling pathways, transcription factors and enzymes at the level of this developmental stage. Reporter ESC lines, flow cytometry and serum-free culture reagents are helping the field to transition from serum-containing protocols to step-wise serum-free differentiation strategies that attempt to mimic the developmental processes in the embryo. This serves as a framework with which to approach directed differentiation of human ESCs for the purposes of regenerative medicine. This review is focused on the contributions that the ESC differentiation system has made to understanding hematopoiesis and will highlight the strengths of this model of development and the challenges it still faces.

Numb mediates the interaction between Wnt and Notch to modulate primitive erythropoietic specification from the hemangioblast.

During embryonic development, the establishment of the primitive erythroid lineage in the yolk sac is a temporally and spatially restricted program that defines the onset of hematopoiesis. In this report, we have used the embryonic stem cell differentiation system to investigate the regulation of primitive erythroid development at the level of the hemangioblast. We show that the combination of Wnt signaling with inhibition of the Notch pathway is required for the development of this lineage. Inhibition of Notch signaling at this stage appears to be mediated by the transient expression of Numb in the hemangioblast-derived blast cell colonies. Activation of the Notch pathway was found to inhibit primitive erythropoiesis efficiently through the upregulation of inhibitors of the Wnt pathway. Together, these findings demonstrate that specification of the primitive erythroid lineage is controlled, in part, by the coordinated interaction of the Wnt and Notch pathways, and position Numb as a key mediator of this process.

Foxo3 is required for the regulation of oxidative stress in erythropoiesis.

Erythroid cells accumulate hemoglobin as they mature and as a result are highly prone to oxidative damage. However, mechanisms of transcriptional control of antioxidant defense in erythroid cells have thus far been poorly characterized. We observed that animals deficient in the forkhead box O3 (Foxo3) transcription factor died rapidly when exposed to erythroid oxidative stress-induced conditions, while wild-type mice showed no decreased viability. In view of this striking finding, we investigated the potential role of Foxo3 in the regulation of ROS in erythropoiesis. Foxo3 expression, nuclear localization, and transcriptional activity were all enhanced during normal erythroid cell maturation. Foxo3-deficient erythrocytes exhibited decreased expression of ROS scavenging enzymes and had a ROS-mediated shortened lifespan and evidence of oxidative damage. Furthermore, loss of Foxo3 induced mitotic arrest in erythroid precursor cells, leading to a significant decrease in the rate of in vivo erythroid maturation. We identified ROS-mediated upregulation of p21(CIP1/WAF1/Sdi1) (also known as Cdkn1a) as a major contributor to the interference with cell cycle progression in Foxo3-deficient erythroid precursor cells. These findings establish an essential nonredundant function for Foxo3 in the regulation of oxidative stress, cell cycle, maturation, and lifespan of erythroid cells. These results may have an impact on the understanding of human disorders in which ROS play a role.

Haemangioblast commitment is initiated in the primitive streak of the mouse embryo.

Haematopoietic and vascular cells are thought to arise from a common progenitor called the haemangioblast. Support for this concept has been provided by embryonic stem (ES) cell differentiation studies that identified the blast colony-forming cell (BL-CFC), a progenitor with both haematopoietic and vascular potential. Using conditions that support the growth of BL-CFCs, we identify comparable progenitors that can form blast cell colonies (displaying haematopoietic and vascular potential) in gastrulating mouse embryos. Cell mixing and limiting dilution analyses provide evidence that these colonies are clonal, indicating that they develop from a progenitor with haemangioblast potential. Embryo-derived haemangioblasts are first detected at the mid-streak stage of gastrulation and peak in number during the neural plate stage. Analysis of embryos carrying complementary DNA of the green fluorescent protein targeted to the brachyury locus demonstrates that the haemangioblast is a subpopulation of mesoderm that co-expresses brachyury (also known as T) and Flk-1 (also known as Kdr). Detailed mapping studies reveal that haemangioblasts are found at highest frequency in the posterior region of the primitive streak, indicating that initial stages of haematopoietic and vascular commitment occur before blood island development in the yolk sac.