Heidi Anderson

Mutation mapping and identification by whole genome sequencing

Genetic mapping of mutations in model systems has facilitated the identification of genes contributing to fundamental biological processes including human diseases. However, this approach has historically required the prior characterization of informative markers. Here we report a fast and cost-effective method for genetic mapping using next-generation sequencing that combines single nucleotide polymorphism discovery, mutation localization, and potential identification of causal sequence variants. In contrast to prior approaches, we have developed a hidden Markov model to narrowly define the mutation area by inferring recombination breakpoints of chromosomes in the mutant pool. In addition, we created an interactive online software resource to facilitate automated analysis of sequencing data and demonstrate its utility in the zebrafish and mouse models. Our novel methodology and online tools will make next-generation sequencing an easily applicable resource for mutation mapping in all model systems.

Cell Surface Structures Influence Lung Clearance Rate of Systemically Infused Mesenchymal Stromal Cells

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM‐MSCs) and umbilical cord blood (UCB‐MSCs). The BM‐MSCs and UCB‐MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB‐MSCs had higher expression levels of α4 integrin (CD49d, VLA‐4), α6 integrin (CD49f, VLA‐6), and the hepatocyte growth factor receptor (c‐Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells. STEM CELLS2013;31:317–326

Functional Network Reconstruction Reveals Somatic Stemness Genetic Maps and Dedifferentiation‐Like Transcriptome Reprogramming Induced by GATA2

Somatic stem cell transplantation holds great promise in regenerative medicine. The best‐characterized adult stem cells are mesenchymal stem cells (MSCs), neural stem cells (NSCs), and CD133+ hematopoietic stem cells (HSCs). The applications of HSCs are hampered since these cells are difficult to maintain in an undifferentiated state in vitro. Understanding genes responsible for stem cell properties and their interactions will help on this issue. The construction of stem cell genetic networks will also help to develop rational strategies to revert somatic cells back to a stem‐like state. We performed a systemic study on human CD133+ HSCs, NSCs, MSCs, and embryonic stem cells and two different progenies of CD133+ HSCs, microvascular endothelial cells (MVECs) and peripheral blood mononuclear cells. Genes abundant in each or in all three somatic stem cells were identified. We also observed complex genetic networks functioning in postnatal stem cells, in which several genes, such as PTPN11 and DHFR, acted as hubs to maintain the stability and connectivity of the whole genetic network. Eighty‐seven HSC genes, including ANGPT1 and GATA2, were independently identified by comparing CD34+CD33−CD38− hematopoietic stem cells with CD34+ precursors and various matured progenies. Introducing GATA2 into MVECs resulted in dedifferentiation‐like transcriptome reprogramming, with HSC genes (such as ANGPT1) being up and endothelial genes (such as EPHB2) being down. This study provides a foundation for a more detailed understanding of human somatic stem cells. Expressing the newly discovered stem cell genes in matured cells might lead to a global reversion of somatic transcriptome to a stem‐like status.

Are globoseries glycosphingolipids SSEA-3 and -4 markers for stem cells derived from human umbilical cord blood?

Umbilical cord blood (UCB) is an efficient and valuable source of hematopoietic stem cells (HSCs) for transplantation. In addition to HSCs it harbours low amounts of mesenchymal stem cells (MSCs). No single marker to identify cord blood-derived stem cells, or to indicate their multipotent phenotype, has been characterized so far. SSEA-3 and -4 are cell surface globoseries glycosphingolipid epitopes that are commonly used as markers for human embryonic stem cells, where SSEA-3 rapidly disappears when the cells start to differentiate. Lately SSEA-3 and -4 have also been observed in MSCs. As there is an ongoing discussion and variation of stem-cell markers between laboratories, we have now comprehensively characterized the expression of these epitopes in both the multipotent stem-cell types derived from UCB. We have performed complementary analysis using gene expression analysis, mass spectrometry and immunochemical methods, including both flow cytometry and immunofluoresence microscopy. SSEA-4, but not SSEA-3, was expressed on MSCs but absent from HSCs. Our findings indicate that SSEA-3 and/or -4 may not be optimal markers for multipotency in the case of stem cells derived from cord blood, as their expression may be altered by cell-culture conditions.

Teleost Growth Factor Independence (Gfi) Genes Differentially Regulate Successive Waves of Hematopoiesis

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.

Human CMP-N-acetylneuraminic acid hydroxylase is a novel stem cell marker linked to stem cell-specific mechanisms.

Human stem cells contain substantial amounts of the xenoantigen N‐glycolylneuraminic acid (Neu5Gc), although the levels of Neu5Gc are low or undetectable in human body fluids and most other human tissues. The lack of Neu5Gc in human tissues has been previously explained by the loss of hydroxylase activity of the human CMP‐N‐acetylneuraminic acid hydroxylase (CMAH) protein caused by a genetic error in the human Cmah gene. We thus wanted to investigate whether the human redundant Cmah gene could still function in stem cell‐specific processes. In this study, we show that CMAH gene expression is significantly upregulated in the adult stem cell populations studied, both of hematopoietic and mesenchymal origin, and identify CMAH as a novel stem cell marker. The CMAH content co‐occurs with higher levels of Neu5Gc within stem cells as measured by mass spectrometric profiling. It seems that despite being enzymatically inactive, human CMAH may upregulate the Neu5Gc content of cells by enhancing Neu5Gc uptake from exogenous sources. Furthermore, exposure to exogenous Neu5Gc caused rapid phosphorylation of β‐catenin in both CMAH overexpressing cells and bone marrow‐derived mesenchymal stem cells, thereby inactivating Wnt/β‐catenin signaling. The data demonstrate the first molecular evidence for xenoantigen Neu5Gc‐induced alteration of crucial stem cell‐specific signaling systems for the maintenance of self renewal. These results add further emphasis to the crucial need for completely xenofree culturing conditions for human stem cells. STEM CELLS 2010;28:258–267

The i Blood Group Antigen as a Marker for Umbilical Cord Blood-Derived Mesenchymal Stem Cells

Multipotent mesenchymal stem cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. However, there is a lack of methods to quickly and efficiently isolate, characterize, and ex vivo expand desired cell populations for therapeutic purposes. Single markers to identify cell populations have not been characterized; instead, all characterizations rely on panels of functional and phenotypical properties. Glycan epitopes can be used for identifying and isolating specific cell types from heterogeneous populations, on the basis of their cell-type specific expression and prominent cell surface localization. We have now studied in detail the cell surface expression of the blood group i epitope (linear poly-N-acetyllactosamine chain) in umbilical cord blood (UCB)-derived MSCs. We used flow cytometry and mass spectrometric glycan analysis and discovered that linear poly-N-acetyllactosamine structures are expressed in UCB-derived MSCs, but not in cells differentiated from them. We further verified the findings by mass spectrometric glycan analysis. Gene expression analysis indicated that the stem-cell specific expression of the i antigen is determined by β3-N-acetylglucosaminyltransferase 5. The i antigen is a ligand for the galectin family of soluble lectins. We found concomitant cell surface expression of galectin-3, which has been reported to mediate the immunosuppressive effects exerted by MSCs. The i antigen may serve as an endogenous ligand for this immunosuppressive agent in the MSC microenvironment. Based on these findings, we suggest that linear poly-N-acetyllactosamine could be used as a novel UCB-MSC marker either alone or within an array of MSC markers.

Hematopoietic stem cells develop in the absence of endothelial cadherin 5 expression

Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from cadherin 5 (Cdh5; vascular endothelial-cadherin)(+) endothelial precursors, and isolated populations of Cdh5(+) cells from mouse embryos and embryonic stem cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Because Cdh5(-/-) mice embryos die before the first HSCs emerge, it is unknown whether Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlb(bw306)), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Using time-lapse confocal imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5(-/-)green fluorescent protein (GFP)(+/+) cells in chimeric mice, we demonstrated that Cdh5(-/-)GFP(+/+) HSCs emerging from embryonic day 10.5 and 11.5 (E10.5 and E11.5) AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multilineage adult blood. We also developed a conditional mouse Cdh5 knockout (Cdh5(flox/flox):Scl-Cre-ER(T)) and demonstrated that multipotent hematopoietic colonies form despite the absence of Cdh5. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs.

Human umbilical cord blood-derived mesenchymal stromal cells display a novel interaction between P-selectin and galectin-1.

Human multipotent mesenchymal stromal/stem cells (MSCs) have been shown to exert immunomodulatory properties that have great potential in therapies for various inflammatory and autoimmune disorders. However, intravenous delivery of these cells is followed by massive cell entrapment in the lungs and insufficient homing to target tissues or organs. In targeting to tissues, MSCs and other therapeutic cells employ similar mechanisms as leucocytes, including a cascade of rolling and adhesion steps mediated by selectins, integrins and their ligands. However, the mechanisms of MSCs homing are not well understood. We discovered that P‐selectin (CD62P) binds to umbilical cord blood (UCB)‐derived MSCs independently of the previously known sialyl Lewis x (sLex)‐containing ligands such as P‐selectin glycoprotein ligand‐1 (PSGL‐1, CD162). By biochemical assays, we identified galectin‐1 as a novel ligand for P‐selectin. Galectin‐1 has previously been shown to be a key mediator of the immunosuppressive effects of human MSCs. We conclude that this novel interaction is likely to play a major role in the immunomodulatory targeting of human UCB‐derived MSCs.

Expression of neural cell adhesion molecule and polysialic acid in human bone marrow-derived mesenchymal stromal cells.

BackgroundIn order to develop novel clinical applications and to gain insights into possible therapeutic mechanisms, detailed molecular characterization of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) is needed. Neural cell adhesion molecule (NCAM, CD56) is a transmembrane glycoprotein modulating cell–cell and cell–matrix interactions. An additional post-translational modification of NCAM is the α2,8-linked polysialic acid (polySia). Because of its background, NCAM is often considered a marker of neural lineage commitment. Generally, hBM-MSCs are considered to be devoid of NCAM expression, but more rigorous characterization is needed.MethodsWe have studied NCAM and polySia expression in five hBM-MSC lines at mRNA and protein levels. Cell surface localization was confirmed by immunofluorescence staining and expression frequency in the donor-specific lines by flow cytometry. For the detection of poorly immunogenic polySia, a fluorochrome-tagged catalytically defective enzyme was employed.ResultsAll five known NCAM isoforms are expressed in these cells at mRNA level and the three main isoforms are present at protein level. Both polysialyltransferases, generally responsible for NCAM polysialylation, are expressed at mRNA level, but only very few cells express polySia at the cell surface.ConclusionsOur results underline the need for a careful control of methods and conditions in the characterization of MSCs. This study shows that, against the generally held view, clinical-grade hBM-MSCs do express NCAM. In contrast, although both polysialyltransferase genes are transcribed in these cells, very few express polySia at the cell surface. NCAM and polySia represent new candidate molecules for influencing MSC interactions.