Martine Geraerts

Upscaling of lentiviral vector production by tangential flow filtration

HIV‐1‐derived vectors are promising tools for gene transfer into the brain. Application of these vectors for gene therapy or for the creation of animal models for neurodegenerative diseases requires standardization and upscaling of lentiviral vector production methods.

Comparison of lentiviral vector titration methods

BackgroundLentiviral vectors are efficient vehicles for stable gene transfer in dividing and non-dividing cells. Several improvements in vector design to increase biosafety and transgene expression, have led to the approval of these vectors for use in clinical studies. Methods are required to analyze the quality of lentiviral vector production, the efficiency of gene transfer and the extent of therapeutic gene expression.ResultsWe compared lentiviral vector titration methods that measure pg p24/ml, RNA equivalents/ml, transducing units (TU/ml) or mRNA equivalents. The amount of genomic RNA in vector particles proves to be reliable to assess the production quality of vectors encoding non-fluorescent proteins. However, the RNA and p24 titers of concentrated vectors are rather poor in predicting transduction efficiency, due to the high variability of vector production based on transient transfection. Moreover, we demonstrate that transgenic mRNA levels correlate well with TU and can be used for functional titration of non-fluorescent transgenes.ConclusionThe different titration methods have specific advantages and disadvantages. Depending on the experimental set-up one titration method should be preferred over the others.

Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm, and Finally Hepatocyte-Like Cells

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development.

Noninvasive and Quantitative Monitoring of Adult Neuronal Stem Cell Migration in Mouse Brain Using Bioluminescence Imaging

It is now generally accepted that continuous neurogenesis occurs in the adult mammalian brain, including that of humans. Modulation of adult neurogenesis can provide therapeutic benefits for various brain disorders, including stroke and Parkinsons disease. The subventricular zone‐olfactory bulb pathway is one of the preferred model systems by which to study neural stem cell proliferation, migration, and differentiation in adult rodent brain. Research on adult neurogenesis would greatly benefit from reliable methods for long‐term noninvasive in vivo monitoring. We have used lentiviral vectors encoding firefly luciferase to stably mark endogenous neural stem cells in the mouse subventricular zone. We show that bioluminescence imaging (BLI) allows quantitative follow‐up of the migration of adult neural stem cells into the olfactory bulb in time. Moreover, we propose a model to fit the kinetic data that allows estimation of migration and survival times of the neural stem cells using in vivo BLI. Long‐term expression of brain‐derived neurotrophic factor in the subventricular zone attenuated neurogenesis, as detected by histology and BLI. In vivo monitoring of the impact of drugs or genes on adult neurogenesis is now within reach.

Novel Hyperactive Transposons for Genetic Modification of Induced Pluripotent and Adult Stem Cells: A Nonviral Paradigm for Coaxed Differentiation

Adult stem cells and induced pluripotent stem cells (iPS) hold great promise for regenerative medicine. The development of robust nonviral approaches for stem cell gene transfer would facilitate functional studies and potential clinical applications. We have previously generated hyperactive transposases derived from Sleeping Beauty, using an in vitro molecular evolution and selection paradigm. We now demonstrate that these hyperactive transposases resulted in superior gene transfer efficiencies and expression in mesenchymal and muscle stem/progenitor cells, consistent with higher expression levels of therapeutically relevant proteins including coagulation factor IX. Their differentiation potential and karyotype was not affected. Moreover, stable transposition could also be achieved in iPS, which retained their ability to differentiate along neuronal, cardiac, and hepatic lineages without causing cytogenetic abnormalities. Most importantly, transposon‐mediated delivery of the myogenic PAX3 transcription factor into iPS coaxed their differentiation into MYOD+ myogenic progenitors and multinucleated myofibers, suggesting that PAX3 may serve as a myogenic “molecular switch” in iPS. Hence, this hyperactive transposon system represents an attractive nonviral gene transfer platform with broad implications for regenerative medicine, cell and gene therapy. STEM CELLS 2010;28:1760–1771

Isolation Procedure and Characterization of Multipotent Adult Progenitor Cells from Rat Bone Marrow

Multipotent adult progenitor cells (MAPCs) are adult stem cells derived from the bone marrow of mouse and rat and were described for the first time in 2002 (Jiang et al., Nature 418:41-49, 2002), and subsequently (Breyer et al., Exp Hematol 34:1596-1601, 2006; Jiang et al., Exp Hematol 30:896-904, 2002; Ulloa-Montoya et al., Genome Biol 8:R163, 2007). The capacity of rodent MAPC to differentiate at the single-cell level into some of the cell types of endoderm, mesoderm, and neuroectoderm germ layer lineages makes them promising candidates for the study of developmental processes. MAPC are isolated using adherent cell cultures and are selected based on morphology after a period of about 8-18 weeks. Here, we describe a step-by-step reproducible method to isolate rat MAPC from fetal and adult bone marrow. We elaborate on several aspects of the isolation protocol including, cell density and medium components, and methods for selecting and obtaining potential MAPC clones and their characterization.

Restoration of Progranulin Expression Rescues Cortical Neuron Generation in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia

Summary To understand how haploinsufficiency of progranulin (PGRN) causes frontotemporal dementia (FTD), we created induced pluripotent stem cells (iPSCs) from patients carrying the GRNIVS1+5G > C mutation (FTD-iPSCs). FTD-iPSCs were fated to cortical neurons, the cells most affected in FTD. Although generation of neuroprogenitors was unaffected, their further differentiation into CTIP2-, FOXP2-, or TBR1-TUJ1 double-positive cortical neurons, but not motorneurons, was significantly decreased in FTD-neural progeny. Zinc finger nuclease-mediated introduction of GRN cDNA into the AAVS1 locus corrected defects in cortical neurogenesis, demonstrating that PGRN haploinsufficiency causes inefficient cortical neuron generation. RNA sequencing analysis confirmed reversal of the altered gene expression profile following genetic correction. We identified the Wnt signaling pathway as one of the top defective pathways in FTD-iPSC-derived neurons, which was reversed following genetic correction. Differentiation of FTD-iPSCs in the presence of a WNT inhibitor mitigated defective corticogenesis. Therefore, we demonstrate that PGRN haploinsufficiency hampers corticogenesis in vitro.

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

Adult stem and progenitor cells.

The discovery of adult stem cells in most adult tissues is the basis of a number of clinical studies that are carried out, with therapeutic use of hematopoietic stem cells as a prime example. Intense scientific debate is still ongoing as to whether adult stem cells may have a greater plasticity than previously thought. Although cells with some features of embryonic stem cells that, among others, express Oct4, Nanog and SSEA1 are isolated from fresh tissue, it is not clear if the greater differentiation potential is acquired during cell culture. Moreover, adult more pluripotent cells do not have all pluripotent characteristics typical for embryonic stem cells. Recently, some elegant studies were published in which adult cells could be completely reprogrammed to embryonic stem cell-like cells by overexpression of some key transcription factors for pluripotency (Oct4, Sox2, Klf4 and c-Myc). It will be interesting for the future to investigate the exact mechanisms underlying this reprogramming and whether similar transcription factor pathways are present and/or can be activated in adult more pluripotent stem cells.