Christoph Schaniel

RAPID AND COORDINATED SWITCH IN CHEMOKINE RECEPTOR EXPRESSION DURING DENDRITIC CELL MATURATION

Dendritic cells (DC) migrate into inflamed peripheral tissues where they capture antigens and, following maturation, to lymph nodes where they stimulate T cells. To gain insight into this process we compared chemokine receptor expression in immature and mature DC. Immature DC expressed CCR1, CCR2, CCR5 and CXCR1 and responded to their respective ligands, which are chemokines produced at inflammatory sites. Following stimulation with LPS or TNF‐α maturing DC expressed high levels of CCR7 mRNA and acquired responsiveness to the CCR7 ligand EBI1 ligand chemokine (ELC), a chemokine produced in lymphoid organs. Maturation also resulted in up‐regulation of CXCR4 and down‐regulation of CXCR1 mRNA, while CCR1 and CCR5 mRNA were only marginally affected for up to 40 h. However, CCR1 and CCR5 were lost from the cell surface within 3 h, due to receptor down‐regulation mediated by chemokines produced by maturing DC. A complete down‐regulation of CCR1 and CCR5 mRNA was observed only after stimulation with CD40 ligand of DC induced to mature by LPS treatment. These different patterns of chemokine receptors are consistent with “inflammatory” and “primary response” phases of DC function.

Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS–mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.

Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells

Directed differentiation of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells captures in vivo developmental pathways for specifying lineages in vitro, thus avoiding perturbation of the genome with exogenous genetic material. Thus far, derivation of endodermal lineages has focused predominantly on hepatocytes, pancreatic endocrine cells and intestinal cells. The ability to differentiate pluripotent cells into anterior foregut endoderm (AFE) derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as the thymus; for metabolism, such as thyroid and parathyroid; and for respiratory function, such as trachea and lung. We find that dual inhibition of transforming growth factor (TGF)-β and bone morphogenic protein (BMP) signaling after specification of definitive endoderm from pluripotent cells results in a highly enriched AFE population that is competent to be patterned along dorsoventral and anteroposterior axes. These findings provide an approach for the generation of AFE derivatives.

Selection events operating at various stages in B cell development

B cells have to progress through various checkpoints during their process of development. The three transcription factors E2A, EBF (early B cell factor) and Pax5 play essential roles in B cell commitment checkpoints. The various forms of the BCR and their downstream signaling molecules, which are expressed at different stages of B cell development, act as critical checkpoint guards allowing (positive selection) or preventing (negative selection) developmental progression. The recent advances on the molecular mechanisms operating at these various checkpoints are here summarized and discussed.

The Role of Chemokines in Regulating Cell Migration during Humoral Immune Responses

Once APC are in the tissues, and resting, mature T and B cells are in their proper areas in the secondary lymphoid organs, the major participants in an immune response are in place to react to the invasion of foreign antigen. However, they remain segregated until they need to cooperate in a humoral response.An immune response to a foreign invader often begins in the epithelia of skin and mucous membranes with which we face the outside world. It appears that native antigen must reach two types of primary target cells: a resting DC and a resting B cell. Binding and uptake of antigen activates both cells, perhaps without the help of other cells. Activation of DC induces the production of proinflammatory chemokines that recruit other myeloid cells and T cells into the area of antigen invasion. At the same time, locally released inflammatory stimuli (e.g., IL-1, TNFα, LPS, double-stranded RNA) induce maturation of immature DC to mature DC. In this process DC switch their chemokine receptor profile and upregulate the expression of CCR7 (Sallusto et al. 1998xSallusto, F, Schaerli, P, Loetscher, P, Schaniel, C, Lenig, D, Mackay, C.R, Qin, S, and Lanzavecchia, A. Eur. J. Immunol. 1998; 28: 2760–2769Crossref | PubMed | Scopus (815)See all ReferencesSallusto et al. 1998). CCR7-expressing DC enter the circulation via blood and lymph and arrive at the entry sites (the HEV) in lymph nodes, and are admitted to the lymphoid organs in an SLC-dependent process (Figure 2Figure 2, step 1; 7xGunn, M.D, Tangemann, K, Tam, C, Cyster, J.G, Rosen, S.D, and Williams, L.T. Proc. Natl. Acad. Sci. USA. 1998; 95: 258–263Crossref | PubMed | Scopus (738)See all References, 8xGunn, M.D, Kyuwa, S, Tam, C, Kakiuchi, T, Matsuzawa, A, Williams, L.T, and Nakano, H. J. Exp. Med. 1999; 189: 451–460Crossref | PubMed | Scopus (776)See all References). ELC produced by previously recruited mature DC then attract the newly arrived DC into the same T cell–DC interaction regions in and near the PALS (Figure 2Figure 2, step 3).In the T cell–rich areas (PALS, paracortex) the mature antigen-presenting DC expressing cell surface antigenic peptide–MHC complexes activate T cells expressing cognate TCR (Figure 2Figure 2, step 4). Forster and colleagues 1999xForster, R, Schubel, A, Breitfeld, D, Kremmer, E, Renner-Muller, I, Wolf, E, and Lipp, M. Cell. 1999; 99: 23–33Abstract | Full Text | Full Text PDF | PubMed | Scopus (1442)See all ReferencesForster and colleagues 1999 have found that in CCR7-deficient mice DC are unable to migrate to secondary lymphoid organs and into the T zones. Hence, primary responses of T cells, both to T cell–dependent antigens and in delayed-type hypersensitivity reactions, are severely reduced or absent. The T cell response takes longer to reach the peak attained in wild-type mice, as if fewer T cells could be initially activated and recruited into the response. However, T cell–dependent responses to antigen become normal with time and appear to be ongoing, as if (T cell–dependent?) downmodulation of humoral responses were impaired.After activation of T cells, helper T cells (TH) develop. Since the activated DC secrete MDC (ABCD-1) and TARC (ABCD-2), these chemokines might be responsible for keeping the activated TH cells attracted to the DC presenting MHC-peptide complexes, so that continuous DC–T cell interaction maintains the stimulation of the TH cells (Figure 2Figure 2, step 5; Schaniel et al. 1999xSchaniel, C, Sallusto, F, Ruedl, C, Sideras, P, Melchers, F, and Rolink, A.G. Eur. J. Immunol. 1999; 29: 2934–2947Crossref | PubMedSee all ReferencesSchaniel et al. 1999). This sets the stage for a T helper cell–dependent B cell response, known to be effective in hypermutation and Ig class switching. It should be stressed that the actions of MDC (ABCD-1) and TARC (ABCD-2) have so far only been demonstrated in vitro, but not in vivo.In parallel, native antigen enters the B cell–rich areas, binds to surface Ig on resting B cells and activates them (Figure 2Figure 2, step 6). This may occur in combination with the costimulatory action of inflammatory molecules, such as LPS and double-stranded nucleic acids, often without the help of T cells and outside the follicular region. Once the B cells are stimulated, all the major participants in the humoral immune response have been activated by foreign antigen, but B cells will either need to come together with the DC–T cell conjugates or recruit activated TH cells. It is not clear whether initially the B cells migrate to the DC–T cell conjugates, either within the T cell–rich areas or at the borders to the B cell areas (Figure 2Figure 2, step 7 opposite direction), or whether the activated T cells migrate to the B cells (Figure 2Figure 2, step 7). The latter direction of migration might be imposed by MDC (ABCD-1) and TARC (ABCD-2) which are also produced by the activated B cells (Schaniel et al. 1999xSchaniel, C, Sallusto, F, Ruedl, C, Sideras, P, Melchers, F, and Rolink, A.G. Eur. J. Immunol. 1999; 29: 2934–2947Crossref | PubMedSee all ReferencesSchaniel et al. 1999). It should be mentioned here that cell migration in vivo most likely involves step-by-step navigation from one gradient to another in complex chemoattractant fields (see Foxman et al. 1997xFoxman, E.F, Campbell, J.J, and Butcher, E.C. J. Cell Biol. 1997; 139: 1349–1360Crossref | PubMed | Scopus (347)See all ReferencesFoxman et al. 1997). Thus, the migration of activated T cells from the DC to the activated B cells probably occurs in such a complex field that likely contains additional chemokines besides MDC (ABCD-1) and TARC (ABCD-2). In the end, the activated B cells are attracted to a fourth type of cell, the follicular dendritic cells (FDC), which are thought to act as depositories of native antigen, as it is presented to sIg on B cells. The FDC form a mesh of structures on which B cells proliferate and form germinal centers (GC). This attraction is mediated by the chemokine BLC, possibly secreted by the FDC, and recognized by CXCR5 expressed on the B cells (Figure 2Figure 2, step 8). There is cross-talk between activated B cells and FDC. The activated B cells must secrete LTα, LTβ, and TNFα that is recognized by the LTβ receptor (LTβ-R) on FDC, before the FDC can differentiate to secrete BLC. Mice deficient in LTα, LTβ, and TNFα and LTβ-R are defective in formation of GC at this stage (Ngo et al. 1999xNgo, V.N, Korner, H, Gunn, M.D, Schmidt, K.N, Sean Riminton, D, Cooper, M.D, Browning, J.L, Sedgwick, J.D, and Cyster, J.G. J. Exp. Med. 1999; 189: 403–412Crossref | PubMed | Scopus (418)See all ReferencesNgo et al. 1999; and references therein), as are mice defective in CXCR5 (Forster et al. 1996xForster, R, Mattis, A.E, Kremmer, E, Wolf, E, Brem, G, and Lipp, M. Cell. 1996; 87: 1037–1047Abstract | Full Text | Full Text PDF | PubMed | Scopus (760)See all ReferencesForster et al. 1996). However, this scenario is likely to be more complex, involving multistep navigation of activated B cells to form GC since Forster et al. 1996xForster, R, Mattis, A.E, Kremmer, E, Wolf, E, Brem, G, and Lipp, M. Cell. 1996; 87: 1037–1047Abstract | Full Text | Full Text PDF | PubMed | Scopus (760)See all ReferencesForster et al. 1996 showed that CXCR5-deficient mice have normal follicles in lymph nodes indicating that CXCR5 cannot be universally required for these events.All together, this cellular trafficking not only brings together the participants of a humoral response, but also selects and topologically separates the antigen-specific antigen-reactive cells from all other resting cells. MDC (ABCD-1) and TARC (ABCD-2) could allow TH cells to cooperate specifically with activated B cells, which then proliferate extensively, hypermutate their Ig V regions and switch their Ig classes in the mesh of FDC (Figure 2Figure 2, step 8). The FDC may also support direct recruitment of, at least, a subset of CD4+ TH cells that expresses CXCR5 (Ansel et al. 1999xAnsel, K.M, McHeyzer-Williams, L.J, Ngo, V.N, McHeyzer-Williams, M.G, and Cyster, J.G. J. Exp. Med. 1999; 190: 1123–1134Crossref | PubMed | Scopus (291)See all ReferencesAnsel et al. 1999). At the end of the response a GC has formed. Memory B cells and plasma cells generated in the GC are thought to emigrate, either to sites of inflammation or to the bone marrow (via lymph and blood) where they can be localized long after the response to foreign antigen has subsided.This review has focused only on one aspect of the molecular and cellular interactions of a humoral immune response: chemokines and their receptors. Cell adhesion is controlled by selectins, transmigration mediated by integrins, cell–cell cooperation by costimulatory molecules and by cytokines. A much more sophisticated program of cellular changes during this response is already partially known, but has not been considered here: e.g., changes during differentiation from immature to mature to memory T and B cells, B1-type and conventional B cell responses, and the involvement of other hematopoietic and nonhematopoietic cells. However, it should be clear from this simplified description of the topology of a humoral response that cell migrations are an indispensable requirement for the proper execution of this response, and that many more modes of attraction are still to be discovered.

MacroH2A histone variants act as a barrier upon reprogramming towards pluripotency

The chromatin template imposes an epigenetic barrier during the process of somatic cell reprogramming. Here, using fibroblasts derived from macroH2A double knockout mice we show that these histone variants act cooperatively as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms, we further demonstrate that macroH2A2 is the predominant barrier to reprogramming. Genomic analyses reveal that macroH2A1 and macroH2A2, together with H3K27me3, co-occupy pluripotency genes in wild type fibroblasts. In particular, we find macroH2A isoforms to be highly enriched at target genes of the K27me3 demethylase, Utx, which are reactivated early in iPS reprogramming. Finally, while macroH2A double knockout induced pluripotent cells are able to differentiate properly in vitro and in vivo, such differentiated cells retain the ability to return to a stem-like state. Therefore, we propose that macroH2A isoforms provide a redundant silencing layer or terminal differentiation ‘lock’ at critical pluripotency genes that presents as an epigenetic barrier when differentiated cells are challenged to reprogram.

Smarcc1/Baf155 Couples Self‐Renewal Gene Repression with Changes in Chromatin Structure in Mouse Embryonic Stem Cells

Little is known about the molecular mechanism(s) governing differentiation decisions in embryonic stem cells (ESCs). To identify factors critical for ESC lineage formation, we carried out a functional genetic screen for factors affecting Nanog promoter activity during mESC differentiation. We report that members of the PBAF chromatin remodeling complex, including Smarca4/Brg1, Smarcb1/Baf47, Smarcc1/Baf155, and Smarce1/Baf57, are required for the repression of Nanog and other self‐renewal gene expression upon mouse ESC (mESC) differentiation. Knockdown of Smarcc1 or Smarce1 suppressed loss of Nanog expression in multiple forms of differentiation. This effect occurred in the absence of self‐renewal factors normally required for Nanog expression (e.g., Oct4), possibly indicating that changes in chromatin structure, rather than loss of self‐renewal gene transcription per se, trigger differentiation. Consistent with this notion, mechanistic studies demonstrated that expression of Smarcc1 is necessary for heterochromatin formation and chromatin compaction during differentiation. Collectively, our data reveal that Smarcc1 plays important roles in facilitating mESCs differentiation by coupling gene repression with global and local changes in chromatin structure. STEM CELLS 2009;27:2979–2991

Zfp281 mediates Nanog autorepression through recruitment of the NuRD complex and inhibits somatic cell reprogramming

The homeodomain transcription factor Nanog plays an important role in embryonic stem cell (ESC) self-renewal and is essential for acquiring ground-state pluripotency during reprogramming. Understanding how Nanog is transcriptionally regulated is important for further dissecting mechanisms of ESC pluripotency and somatic cell reprogramming. Here, we report that Nanog is subjected to a negative autoregulatory mechanism, i.e., autorepression, in ESCs, and that such autorepression requires the coordinated action of the Nanog partner and transcriptional repressor Zfp281. Mechanistically, Zfp281 recruits the NuRD repressor complex onto the Nanog locus and maintains its integrity to mediate Nanog autorepression and, functionally, Zfp281-mediated Nanog autorepression presents a roadblock to efficient somatic cell reprogramming. Our results identify a unique transcriptional regulatory mode of Nanog gene expression and shed light into the mechanistic understanding of Nanog function in pluripotency and reprogramming.

Modeling Familial Cancer with Induced Pluripotent Stem Cells

In vitro modeling of human disease has recently become feasible with induced pluripotent stem cell (iPSC) technology. Here, we established patient-derived iPSCs from a Li-Fraumeni syndrome (LFS) family and investigated the role of mutant p53 in the development of osteosarcoma (OS). LFS iPSC-derived osteoblasts (OBs) recapitulated OS features including defective osteoblastic differentiation as well as tumorigenic ability. Systematic analyses revealed that the expression of genes enriched in LFS-derived OBs strongly correlated with decreased time to tumor recurrence and poor patient survival. Furthermore, LFS OBs exhibited impaired upregulation of the imprinted gene H19 during osteogenesis. Restoration of H19 expression in LFS OBs facilitated osteoblastic differentiation and repressed tumorigenic potential. By integrating human imprinted gene network (IGN) into functional genomic analyses, we found that H19 mediates suppression of LFS-associated OS through the IGN component DECORIN (DCN). In summary, these findings demonstrate the feasibility of studying inherited human cancer syndromes with iPSCs.

Epigenetic reprogramming induces the expansion of cord blood stem cells

Cord blood (CB) cells that express CD34 have extensive hematopoietic capacity and rapidly divide ex vivo in the presence of cytokine combinations; however, many of these CB CD34+ cells lose their marrow-repopulating potential. To overcome this decline in function, we treated dividing CB CD34+ cells ex vivo with several histone deacetylase inhibitors (HDACIs). Treatment of CB CD34+ cells with the most active HDACI, valproic acid (VPA), following an initial 16-hour cytokine priming, increased the number of multipotent cells (CD34+CD90+) generated; however, the degree of expansion was substantially greater in the presence of both VPA and cytokines for a full 7 days. Treated CD34+ cells were characterized based on the upregulation of pluripotency genes, increased aldehyde dehydrogenase activity, and enhanced expression of CD90, c-Kit (CD117), integrin α6 (CD49f), and CXCR4 (CD184). Furthermore, siRNA-mediated inhibition of pluripotency gene expression reduced the generation of CD34+CD90+ cells by 89%. Compared with CB CD34+ cells, VPA-treated CD34+ cells produced a greater number of SCID-repopulating cells and established multilineage hematopoiesis in primary and secondary immune-deficient recipient mice. These data indicate that dividing CB CD34+ cells can be epigenetically reprogrammed by treatment with VPA so as to generate greater numbers of functional CB stem cells for use as transplantation grafts.