An ongoing production of IFN-α may be of etiopathogenic significance in systemic lupus erythematosus (SLE). It may be due to the natural IFN-producing cells (NIPC), also termed plasmacytoid dendritic cells (PDC), activated by immune complexes that contain nucleic acids derived from apoptotic cells. We here examined the role of FcγR in the IFN-α production in vitro by PBMC induced by the combination of apoptotic U937 cells and autoantibody-containing IgG from SLE patients (SLE-IgG). The Fc portion of the SLE-IgG was essential to induce IFN-α production, because Fab fragments or F(ab′)2 were ineffective. Normal, especially heat-aggregated, IgG inhibited the IFN-α production, suggesting a role for FcγR on PBMC. Using blocking anti-FcγR Abs, the FcγRIIa,c (CD32) but not FcγRI or FcγRIII were shown to be involved in the IFN-α induction by apoptotic cells combined with SLE-IgG, but not by HSV or CpG DNA. In contrast, the action of all of these inducers was inhibited by the anti-FcγRIIa,b,c mAb AT10 or heat-aggregated IgG. Flow cytometric analysis revealed that ∼50% of the BDCA-2-positive PBMC, i.e., NIPC/PDC, expressed low but significant levels of FcγRII, as did most of the actual IFN-α producers activated by HSV. RT-PCR applied to NIPC/PDC purified by FACS demonstrated expression of FcγRIIa, but not of FcγRIIb or FcγRIIc. We conclude that FcγRIIa on NIPC/PDC is involved in the activation of IFN-α production by interferogenic immune complexes, but may also mediate inhibitory signals. The FcγRIIa could therefore have a key function in NIPC/PDC and be a potential therapeutic target in SLE.

The derivation of germ cells from human embryonic stem cells (hESCs) or human induced pluripotent stem (hIPS) cells represents a desirable experimental model and potential strategy for treating infertility. In the current study we developed a triple biomarker assay for identifying and isolating human primordial germ cells (PGCs) by first evaluating human PGC formation during the first trimester in vivo. Next, we applied this technology to characterizing in vitro derived PGCs (iPGCs) from pluripotent cells. Our results show that co-differentiation of hESCs on human fetal gonadal stromal cells significantly improves the efficiency of generating iPGCs. Furthermore, the efficiency was comparable between various pluripotent cell lines regardless of origin from the inner cell mass of human blastocysts (hESCs), or reprogramming of human skin fibroblasts (hIPS). In order to better characterize the iPGCs we performed Real time PCR, microarray and bisulfite sequencing. Our results show that iPGCs at day 7 of differentiation are transcriptionally distinct from the somatic cells, expressing genes associated with pluripotency and germ cell development while repressing genes associated with somatic differentiation (specifically multiple HOX genes). Using bisulfite sequencing, we show that iPGCs initiate imprint erasure from differentially methylated imprinted regions by day 7 of differentiation. However, iPGCs derived from hIPS cells do not initiate imprint erasure as efficiently. In conclusion, our results indicate that triple positive iPGCs derived from pluripotent cells differentiated on hFGS cells correspond to committed first trimester germ cells (before 9 weeks) that have initiated the process of imprint erasure.

Enforced expression of IntroductionHematopoiesis relies on the unique abilities of relatively few hematopoietic stem cells to self-renew and generate progenitors that will differentiate into the mature cells forming the blood system. This dynamic process is tightly regulated by a complex of internal and external signals, such as transcription factors, growth factors, and cell cycle regulators (for reviews, see Orkin 1 and Verfaillie 2 ). Many transcription factors, including homeobox (Hox) transcription factors, have been shown to be key players in the proliferation and differentiation of early progenitor cells. 3,[4][5][6] Specific expression patterns of multiple Hox genes have been detected in normal and leukemic hematopoiesis. 7,8 Enforced expression of Hox genes has been shown to affect the ability of progenitors and stem cells to proliferate and differentiate. [9][10][11][12][13][14][15][16][17] One of these genes, Hoxb4, has been implicated in the regulation of hematopoietic stem cell regeneration, 8 and retrovirally engineered overexpression in murine bone marrow cells dramatically increases the stem cell pool ex vivo and in vivo, resulting in faster, more complete recovery of the stem cells in transplantation studies with no adverse effect on differentiation or lineage distribution. 14,[18][19][20][21] This is in contrast to the overexpression of other Hox genes, which can perturb the proliferation and lineage commitment of primitive progenitors and can give rise to hematopoietic malignancies. 10,11,13,15,16,[22][23][24] However, recent studies have suggested that the effect of Hoxb4 is concentration dependent and is not necessarily restricted to proliferation. [25][26][27] Thus, the level of Hoxb4 expression has to be within a specific range for Hoxb4 to increase stem cell proliferation without adverse effects on differentiation. Although enforced expression of Hoxb4 in hematopoietic cells has been studied in detail, its physiologic role in hematopoiesis is poorly understood. Recently, we described a mouse model deficient in Hoxb3 and Hoxb4, showing reduced proliferative capacity of the stem cell pool without otherwise perturbing hematopoiesis. 28 Here we report a novel mouse model in which the Hoxb4 gene alone has been completely removed through the Cre/loxP technique. Hoxb4-deficient mice have a phenotype similar to that of double Hoxb3/Hoxb4 knockout (KO) mice, although the effects are milder in the Hoxb4 Ϫ/Ϫ mice. The phenotype observed seems mainly confined to the stem cell pool, resulting in reduced proliferative capacity of bone marrow and fetal liver hematopoietic stem cells (HSCs) without affecting differentiation or lineage choice. Deficiency of Hoxb4 or Hoxb3 and Hoxb4 affects the expression of other Hox genes and the expression of cell cycle regulators, indicating a complex regulatory role of these Hox genes. Collectively, these findings indicate that Hoxb4 improves proliferative recruitment of HSCs in settings demanding high proliferation, such as transplantation, but that it has a less pro...

Limited knowledge of the mechanisms that govern the self-renewal of human haematopoietic stem cells (HSCs), and why this fails in culture, have impeded the expansion of HSCs for transplantation 1 . Here we identify MLLT3 (also known as AF9) as a crucial regulator of HSCs that is highly enriched in human fetal, neonatal and adult HSCs, but downregulated in culture. Depletion of MLLT3 prevented the maintenance of transplantable human haematopoietic stem or Reprints and permissions information is available at http://www.nature.com/reprints.