Annelies Crabbe

Effects of MRI Contrast Agents on the Stem Cell Phenotype

The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival, and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell-based therapies. To follow the fate of grafted cells in vivo, a number of noninvasive imaging approaches have been developed. Magnetic resonance imaging (MRI) is a high-resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of three different stem cell populations [mouse embryonic stem cells (mESC), rat multipotent adult progenitor cells (rMAPC), and mouse mesenchymal stem cells (mMSC)] with three different (ultra)small superparamagnetic iron oxide [(U)SPIO] particles (Resovist®, Endorem®, Sinerem®) was compared. Labeling efficiency with Resovist® and Endorem® differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI-based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.

MAPC culture conditions support the derivation of cells with nascent hypoblast features from bone marrow and blastocysts

Dear Editor, We previously demonstrated (Jiang et al., 2002) that rodent multipotent adult progenitor cells (MAPC) can self-renew longterm while maintaining multilineage differentiation capacity. Rodent MAPC express a number of pluripotency-related transcription factors (TF) including Oct4 and Rex1 but not Nanog and Sox2, two other TF known to play a significant role in the maintenance of the pluripotency of embryonic stem cells (ESC) (Ulloa-Montoya et al., 2007). However, rodent MAPC express several TF, including Gata4, Gata6, Sox7 and Sox17, typically expressed in the nascent hypoblast of the developing inner cell mass (ICM) (Nichols and Smith, 2011) and in the recently described rat extrambryonic endodermal precursor cells (rXEN-P), which are isolated from blastocyst (Debeb et al., 2009). We derived in 4/12 independent isolations one or more rMAPC lines, by culturing rat BM cells in rMAPC medium (rMAPC isolation scheme, Supplementary Figure S1). After 4 weeks of culture, BM cells were depleted of CD45+ cells and 2–8 weeks later, clusters of refractile and small cells appeared, which became the preponderant cell type within 10 days (Figure 1A). Nearly all cells from the established lines expressed Oct4, Gata4, Gata6, Sox7 and Sox17 transcripts and proteins (Figure 1B and Supplementary Figure S2A and B), as well the surface markers SSEA1 and CD31 (Figure 1C and Supplementary Figure S2C), both markers of the early ICM. Although rMAPC lines express Oct4, previous studies (Lengner et al., 2007) demonstrated that Oct4+ cells cannot be detected in adult mouse tissues and that Oct4 is not required for postnatal tissue homeostasis. Based on Lengner’s findings, we hypothesized that the rMAPC phenotype could be the result of a culture-induced reprogramming. We therefore analysed BM-cultures during 2 independent rMAPC isolations before, during and after the appearance of the refractile and small cells. We could not identify any Oct4+ or SSEA1+/CD31+ cells in more than one million cells analyzed after CD45+ cells depletion (Figure 1C and D, and Supplementary Figure S2D and E), several weeks before the appearance of the refractile cells positive for these markers. RT-qPCR analysis further demonstrated that acquisition of the typical rMAPC morphology was associated with .1000fold increase in expression of Oct4 and the typical hypoblast gene transcripts (Figure 1E and Supplementary Figure S2F). Although some rMAPC lines had karyotypical abnormalities (Supplementary Figure S2G and Table S1), some lines did not, suggesting that the rMAPC phenotype is not induced by a specific translocation, duplication and/or deletion. These studies demonstrate that rMAPC do not exist in BM and that this hypoblast phenotype is acquired upon prolonged in vitro culture. rMAPC may represent a rare event of in vitro reprogramming, resembling what has been observed during spermatogonial stem cell (Guan et al., 2006; Kanatsu-Shinohara et al., 2008; Ko et al., 2009) and epiblast stem cell (Bao et al., 2009) de-differentiation to ESC-like cells, when cultured under ESC conditions. Because the gene expression pattern of rMAPC (Ulloa-Montoya et al., 2007) and rXEN-P cells (Debeb et al., 2009) is highly similar, we next asked whether BM cells were reprogrammed to a hypoblast/extraembryonic progenitor fate. To investigate this, we tested whether rMAPC could be cultured under rXEN-P conditions and vice versa. When established rXEN-P lines were cultured under rMAPC conditions for 1–2 passages, they grew dispersed, acquiring the typical rMAPC morphology (Supplementary Figure S3A). rXEN-P cells became homogeneously Oct4+/Gata4+ and the percentage of SSEA1+ cells increased (Supplementary Figure S3B and E). RT-qPCR revealed that no differences in RNA expression for hypoblast genes could be detected in XEN-P lines, once cultured in MAPC conditions, except for higher levels of Sox17 and lower levels of Tmprss2 (Supplementary Figure S3D). By contrast, when rMAPC were cultured in XEN-P medium on rat embryonic feeders, typical XEN-P colonies were generated, i.e. Oct42/Gata4+ epithelioid cells with a rim of loosely attached small refractile cells that are Oct4+/Gata4+ (Supplementary Figure S3F and G). Moreover, the percentage of SSEA1+ cells decreased (Supplementary Figure S3J); consistently, RT-qPCR revealed a decrease in Sox17 and an increase in Tmprss2 (Supplementary Figure S3I). Cell doubling time of rMAPC or rXEN-P cells cultured in MAPC conditions was slightly faster than in XEN-P conditions (Supplementary Figure S3A and F). Therefore, rMAPC culture conditions supported the feederfree growth of established XEN-P clones in a more homogenous and immature state. To further define the relationship between rMAPC, XEN-P and typical XEN cells, rMAPC and rXEN-P cells were also cultured under standard XEN conditions (Kunath et al., 2005) without exogenous LIF (Supplementary Figure S4A). rXEN-P and rMAPC cells in XEN conditions, formed extraembryonic endodermal colonies with significantly lower proliferation rate (Supplementary Figure S4B and E). Expression of hypoblast gene transcripts doi:10.1093/jmcb/mjs046 Journal of Molecular Cell Biology (2012), 4, 423–426 | 423 Published online August 9, 2012

PEGylation of phospholipids improves their intermembrane exchange ratePresented at the 17th Conference of the European Colloid & Interface Science Society, Firenze, Italy, September 21?26, 2003.

The polar headgroup of dimyristoylphosphatidylethanolamine (DMPE) was modified by attaching a hydrophilic polymer-amino acid complex and the effects of this modification on the non-protein-mediated transfer features of the lipid determined. The first step in the organic synthesis protocol was the conversion of α,ω-dicarboxy poly(ethylene glycol) into its anhydride form, followed by attachment of the molecule to the amino group of DMPE. In the second step, the remaining free –COOH group on the polymer terminus was activated consecutively with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sulfo-N-hydroxysuccinimide, and then coupled to tryptophan. The purified conjugate was mixed with dipentadecanoylphosphatidylglycerol (1/9 molar ratio) and sonicated to produce small unilamellar vesicles. These vesicles (donors) were then mixed with dipentadecanoylphosphatidylglycerol magnetoliposomes (acceptors) and the transfer kinetics of the modified lipid followed, using high-gradient magnetophoresis as the fractionation technique. The transfer behavior of non-modified DMPE was also examined. The first-order kinetic plots, corrected for back exchange lipid movement, showed that hydrophilization of DMPE dramatically improved its transfer capacity. These findings are explained by considering the thermodynamical consequences of the exchange process. The possibility of using biomolecule-derivatized phospholipids to trigger physiological effects in biological cells is briefly discussed.

Chapter 28 – Multipotent Adult Progenitor Cells

Embryonic stem cells (ESCs) are pluripotent stem cells as they can be propagated indefinitely and differentiate into cells of all three germ layers (endoderm, mesoderm, and ectoderm), shown by teratoma and embryoid body (EB) formation. ESCs are derived from the inner cell mass (ICM) of the blastocyst and are true pluripotent stem cells. Mouse ESCs express the cell surface antigen SSEA1 and human ESC SSEA4, and both are characterized by the expression of a number of relative ESC-specific genes, including the transcription factors (TFs) Oct4, Rex1, Nanog, and Sox2. Forced expression of Nanog in ESC results in LIF-independent proliferation, demonstrating its important role in maintaining ESC pluripotency. The pluripotent cells in the ICM become restricted first to a specific germ layer and then to a specific tissue during gastrulation. The latter persist throughout adult life and are termed multipotent stem cells. The mouse adult fibroblasts can be reprogrammed towards cells with all ESC characteristics, so-called induced pluripotent stem cells (iPSCs), by the introduction of four transcription factors known to be expressed in ESCs ( Oct4, Sox2, Klf4, and c-Myc ) and selecting for cells that start to express endogenous Nanog or Oct4 . Transfection with Oct4, Sox2, and Klf4 drives somatic cells to a Nanog - pre-pluripotent stage and acquisition of Nanog is mandatory to gain full reprogramming to pluripotent cells. This provides proof of the principle that adult somatic cells can be reprogrammed.