Niels-Bjarne Woods

Hematopoietic Cell-Specific Deletion of Toll-like Receptor 4 Ameliorates Hepatic and Adipose Tissue Insulin Resistance in High-Fat-Fed Mice

Chronic low-grade inflammation, particularly in adipose tissue, is an important modulator of obesity-induced insulin resistance. The Toll-like receptor 4 (Tlr4) is a key initiator of inflammatory responses in macrophages. We performed bone marrow transplantation (BMT) of Tlr4lps-del or control C57Bl/10J donor cells into irradiated wild-type C57Bl6 recipient mice to generate hematopoietic cell-specific Tlr4 deletion mutant (BMT-Tlr4(-/-)) and control (BMT-WT) mice. After 16 weeks of a high-fat diet (HFD), BMT-WT mice developed obesity, hyperinsulinemia, glucose intolerance, and insulin resistance. In contrast, BMT-Tlr4(-/-) mice became obese but did not develop fasting hyperinsulinemia and had improved hepatic and adipose insulin sensitivity during euglycemic clamp studies, compared to HFD BMT-WT controls. HFD BMT-Tlr4(-/-) mice also showed markedly reduced adipose tissue inflammatory markers and macrophage content. In summary, our results indicate that Tlr4 signaling in hematopoietic-derived cells is important for the development of hepatic and adipose tissue insulin resistance in obese mice.

Repopulation of adult and neonatal mice with human hepatocytes: A chimeric animal model

We report the successful transplantation of human hepatocytes in immunodeficient, fumarylacetoacetate hydrolase-deficient (fah−/−) mice. Engraftment occurs over the entire liver acinus upon transplantation. A few weeks after transplantation, increasing concentrations of human proteins (e.g., human albumin and human C3a) can be measured in the blood of the recipient mouse. No fusion between mouse and human hepatocytes can be detected. Three months after transplantation, up to 20% of the mouse liver is repopulated by human hepatocytes, and sustained expression of lentiviral vector transduced gene can be observed. We further report the development of a hepatocyte transplantation method involving a transcutaneous, intrahepatic injection in neonatal mice. Human hepatocytes engraft over the entire injected lobe with an expansion pattern similar to those observed with intrasplenic transplantation.

Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro.

ABSTRACT The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.

Genetic Variability Overrides the Impact of Parental Cell Type and Determines iPSC Differentiation Potential

Summary Reports on the retention of somatic cell memory in induced pluripotent stem cells (iPSCs) have complicated the selection of the optimal cell type for the generation of iPSC biobanks. To address this issue we compared transcriptomic, epigenetic, and differentiation propensities of genetically matched human iPSCs derived from fibroblasts and blood, two tissues of the most practical relevance for biobanking. Our results show that iPSC lines derived from the same donor are highly similar to each other. However, genetic variation imparts a donor-specific expression and methylation profile in reprogrammed cells that leads to variable functional capacities of iPSC lines. Our results suggest that integration-free, bona fide iPSC lines from fibroblasts and blood can be combined in repositories to form biobanks. Due to the impact of genetic variation on iPSC differentiation, biobanks should contain cells from large numbers of donors.

Brief Report: Efficient Generation of Hematopoietic Precursors and Progenitors from Human Pluripotent Stem Cell Lines

By mimicking embryonic development of the hematopoietic system, we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines, extra cellular matrix components, and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% ± 16%, from seven pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38− hematopoietic progenitors. Moreover, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays, were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors, it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long‐term repopulating ability. STEM CELLS 2011;29:1158–1164

Lentivirus Gene Transfer in Murine Hematopoietic Progenitor Cells Is Compromised by a Delay in Proviral Integration and Results in Transduction Mosaicism and Heterogeneous Gene Expression in Progeny Cells

ABSTRACT Human immunodeficiency virus type 1-based lentivirus vectors containing the green fluorescent protein (GFP) gene were used to transduce murine Lin− c-kit+ Sca1+primitive hematopoietic progenitor cells. Following transduction, the cells were plated into hematopoietic progenitor cell assays in methylcellulose and the colonies were scored for GFP positivity. After incubation for 20 h, lentivirus vectors transduced 27.3% ± 6.7% of the colonies derived from unstimulated target cells, but transduction was more efficient when the cells were supported with stem cell factor (SCF) alone (42.0% ± 5.5%) or SCF, interleukin-3 (IL-3), and IL-6 (53.3 ± 1.8%) during transduction. The, vesicular stomatitis virus glycoprotein-pseudotyped MGIN oncoretrovirus control vector required IL-3, IL-6, and SCF for significant transduction (39.3 ± 9.4%). Interestingly, only a portion of the progeny cells within the lentivirus-transduced methylcellulose colonies expressed GFP, in contrast to the homogeneous expression in oncoretrovirus-transduced colonies. Secondary plating of the primary GFP+ lentivirus vector-transduced colonies revealed vector PCR+ GFP+ (42%), vector PCR−GFP− (46%), and vector PCR+ GFP−(13%) secondary colonies, indicating true genetic mosaicism with respect to the viral genome in the progeny cells. The degree of vector mosaicism in individual colonies could be reduced by extending the culture time after transduction and before plating into the clonal progenitor cell assay, indicating a delay in the lentiviral integration process. Furthermore, supplementation with exogenous deoxynucleoside triphosphates during transduction decreased mosaicism within the colonies. Although cytokine stimulation during transduction correlates with higher transduction efficiency, rapid cell division after transduction may result in loss of the viral genome in the progeny cells. Therefore, optimal transduction may require activation without promoting intense cell proliferation prior to vector integration.

Development of gene therapy for hematopoietic stem cells using lentiviral vectors.

Lentiviral vectors are promising tools for the development of gene therapy since they can transduce both quiescent and dividing target cells. Lentiviral vectors may be particularly promising gene delivery tools for hematopoietic stem cells since these target cells tend to be quiescent and are therefore difficult target cells for vectors that require dividing targets. Human hematopoietic stem cells that can repopulate NOD/SCID mice have been efficiently transduced using HIV-1-based lentiviral vectors and similar vectors can also transduce murine hematopoietic stem cells. HIV-1 vectors that contain strong general promoters can generate high levels of transgene expression and very high expression levels can be generated in erythroid cells in vivo using β-globin regulatory sequences to control the expression of the transgene. Current lentiviral vectors have a similar level of biosafety as oncoretroviral vectors and can therefore theoretically be used in clinical gene therapy protocols. Future challenges include the generation of lentiviral vectors that can express more than one transgene at high levels and the generation of safe permanent packaging cells for practical use in clinical gene therapy trials.

Retinoic Acid Regulates Hematopoietic Development from Human Pluripotent Stem Cells

Summary The functions of retinoic acid (RA), a potent morphogen with crucial roles in embryogenesis including developmental hematopoiesis, have not been thoroughly investigated in the human setting. Using an in vitro model of human hematopoietic development, we evaluated the effects of RA signaling on the development of blood and on generated hematopoietic progenitors. Decreased RA signaling increases the generation of cells with a hematopoietic stem cell (HSC)-like phenotype, capable of differentiation into myeloid and lymphoid lineages, through two separate mechanisms: by increasing the commitment of pluripotent stem cells toward the hematopoietic lineage during the developmental process and by decreasing the differentiation of generated blood progenitors. Our results demonstrate that controlled low-level RA signaling is a requirement in human blood development, and we propose a new interpretation of RA as a regulatory factor, where appropriate control of RA signaling enables increased generation of hematopoietic progenitor cells from pluripotent stem cells in vitro.

Somatic cells with a heavy mitochondrial DNA mutational load render induced pluripotent stem cells with distinct differentiation defects.

It has become increasingly clear that several age‐associated pathologies associate with mutations in the mitochondrial genome. Experimental modeling of such events has revealed that acquisition of mitochondrial DNA (mtDNA) damage can impair respiratory function and, as a consequence, can lead to widespread decline in cellular function. This includes premature aging syndromes. By taking advantage of a mutator mouse model with an error‐prone mtDNA polymerase, we here investigated the impact of an established mtDNA mutational load with regards to the generation, maintenance, and differentiation of induced pluripotent stem (iPS) cells. We demonstrate that somatic cells with a heavy mtDNA mutation burden were amenable for reprogramming into iPS cells. However, mutator iPS cells displayed delayed proliferation kinetics and harbored extensive differentiation defects. While mutator iPS cells had normal ATP levels and glycolytic activity, the induction of differentiation coincided with drastic decreases in ATP production and a hyperactive glycolysis. These data demonstrate the differential requirements of mitochondrial integrity for pluripotent stem cell self‐renewal versus differentiation and highlight the relevance of assessing the mitochondrial genome when aiming to generate iPS cells with robust differentiation potential. Stem Cells 2014;32:1173–1182

Single-Cell Analysis Identifies Distinct Stages of Human Endothelial-to-Hematopoietic Transition

During development, hematopoietic cells originate from endothelium in a process known as endothelial-to-hematopoietic transition (EHT). To study human EHT, we coupled flow cytometry and single-cell transcriptional analyses of human pluripotent stem cell-derived CD34+ cells. The resulting transcriptional hierarchy showed a continuum of endothelial and hematopoietic signatures. At the interface of these two signatures, a unique group of cells displayed both an endothelial signature and high levels of key hematopoietic stem cell-associated genes. This interphase group was validated via sort and subculture as an immediate precursor to hematopoietic cells. Differential expression analyses further divided this population into subgroups, which, upon subculture, showed distinct hematopoietic lineage differentiation potentials. We therefore propose that immediate precursors to hematopoietic cells already have their hematopoietic lineage restrictions defined prior to complete downregulation of the endothelial signature. These findings increase our understanding of the processes of de novo hematopoietic cell generation in the human developmental context.