STEM CELLS current issue

Concise Review: Adult Multipotent Stromal Cells and Cancer: Risk or Benefit?

Lazennec, G., Jorgensen, C. — 2008-06-12

This review focuses on the interaction between multipotent stromal cells (MSCs) and carcinoma and the possible use of MSCs in cell-based anticancer therapies. MSCs are present in multiple tissues and are defined as cells displaying the ability to differentiate in multiple lineages, including chondrocytes, osteoblasts, and adipocytes. Recent evidence also suggests that they could play a role in the progression of carcinogenesis and that MSCs could migrate toward primary tumors and metastatic sites. It is possible that MSCs could also be involved in the early stages of carcinogenesis through spontaneous transformation. In addition, it is thought that MSCs can modulate tumor growth and metastasis, although this issue remains controversial and not well understood. The immunosuppressive properties and proangiogenic properties of MSCs account, at least in part, for their effects on cancer development. On the other hand, cancer cells also have the ability to enhance MSC migration. This complex dialog between MSCs and cancer cells is certainly critical for the outcome of tumor development. Interestingly, several studies have shown that MSCs engineered to express antitumor factors could be an innovative choice as a cell-mediated gene therapy to counteract tumor growth. More evidence will be needed to understand how MSCs positively or negatively modulate carcinogenesis and to evaluate the safety of MSC use in cell-mediated gene strategies.

Disclosure of potential conflicts of interest is found at the end of this article.

Local Delivery of Granulocyte Colony Stimulating Factor-Mobilized CD34-Positive Progenitor Cells Using Bioscaffold for Modality of Unhealing Bone Fracture

2008-06-12

We recently reported that i.v. transplantation of adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, contributes to fracture healing through the enhancement of vasculogenesis and osteogenesis. However, the scarcity of CD34+ cells in the adult human is a critical issue for the future clinical application of this method. To overcome this issue, we assessed in vitro and in vivo capacity of granulocyte colony-stimulating factor-mobilized peripheral blood (GM-PB) human CD34+ cells for vasculogenesis and osteogenesis. First, we confirmed the differentiation capability of GM-PB CD34+ cells into osteoblasts in vitro. Second, local transplantation of GM-PB CD34+ cells on atelocollagen scaffold was performed in nude rats in a model of unhealing fractures. Immunostaining for human leukocyte antigen-ABC of tissue samples 1 week after fracture and cell therapy showed the superior incorporation after local transplantation compared with systemic infusion. Third, the effects of local transplantation of 10⁵ (Hi), 10⁴ (Mid), or 10³ (Lo) doses of GM-PB CD34+ cells or phosphate-buffered saline (PBS) on fracture healing were compared. Extrinsic vasculogenic and osteogenic differentiation of GM-PB CD34+ cells, enhancement of the intrinsic angio-osteogenesis by recipient cells, augmentation of blood flow recovery at the fracture sites, and radiological and histological confirmation of fracture healing were observed only in the Hi and Mid groups but not in the Lo and PBS groups. These results strongly suggest that local transplantation of GM-PB CD34+ cells with atelocollagen scaffold is a feasible strategy for therapeutic vasculogenesis and osteogenesis needed for fracture healing.

Disclosure of potential conflicts of interest is found at the end of this article.

Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor Mediate Human Stem Cell Tropism to Malignant Solid Tumors

2008-06-12

Human neural and mesenchymal stem cells have been identified for cell-based therapies in regenerative medicine and as vehicles for delivering therapeutic agents to areas of injury and tumors. However, the signals required for homing and recruitment of stem cells to these sites are not well understood. Urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) are involved in chemotaxis and cell guidance during normal development and are upregulated in invasive tumors. Here we provided evidence that activation of uPA and uPAR in malignant solid tumors (brain, lung, prostate, and breast) augments neural and mesenchymal stem cell tropism. Expression levels of uPAR on human solid tumor cell lines correlated with levels of uPA and soluble uPAR in tumor cell-conditioned media. Cytokine expression profiles of these tumor-conditioned media were determined by protein arrays. Among 79 cytokines investigated, interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 were the most highly expressed cytokines in uPAR-positive tumors. We provided evidence that human recombinant uPA induced stem cell migration, whereas depletion of uPA from PC-3 prostate cancer cell-conditioned medium blocked stem cell migration. Furthermore, retrovirus-mediated overexpression of uPA and uPAR in neuroblastoma (NB1691) cells induced robust migration of stem cells toward NB1691 cell-conditioned media, compared with media derived from wild-type NB1691 cells. We conclude that expression of uPA and uPAR in cancer cells underlies a novel mechanism of stem cell tropism to malignant solid tumors, which may be important for development of optimal stem cell-based therapies.

Disclosure of potential conflicts of interest is found at the end of this article.

Direct Orthotopic Transplantation of Fresh Surgical Specimen Preserves CD133+ Tumor Cells in Clinically Relevant Mouse Models of Medulloblastoma and Glioma

2008-06-12

Recent identification of cancer stem cells in medulloblastoma (MB) and high-grade glioma has stimulated an urgent need for animal models that will not only replicate the biology of these tumors, but also preserve their cancer stem cell pool. We hypothesize that direct injection of fresh surgical specimen of MB and high-grade glioma tissues into anatomically equivalent locations in immune-deficient mouse brains will facilitate the formation of clinically accurate xenograft tumors by allowing brain tumor stem cells, together with their non-stem tumor and stromal cells, to grow in a microenvironment that is the closest to human brains. Eight of the 14 MBs (57.1%) and two of the three high-grade gliomas (66.7%) in this study developed transplantable (up to 12 passages) xenografts in mouse cerebellum and cerebrum, respectively. These xenografts are patient specific, replicating the histopathologic, immunophenotypic, invasive/metastatic, and major genetic (analyzed with 10K single nucleotide polymorphism array) abnormalities of the original tumors. The xenograft tumor cells have also been successfully cryopreserved for long-term preservation of tumorigenicity, ensuring a sustained supply of the animal models. More importantly, the CD133⁺ tumor cells, ranging from 0.2%–10.4%, were preserved in all the xenograft models following repeated orthotopic subtransplantations in vivo. The isolated CD133⁺ tumor cells formed neurospheres and displayed multi-lineage differentiation capabilities in vitro. In summary, our study demonstrates that direct orthotopic transplantation of fresh primary tumor cells is a powerful approach in developing novel clinical relevant animal models that can reliably preserve CD133⁺ tumor cell pools even during serial in vivo subtransplantations.

Disclosure of potential conflicts of interest is found at the end of this article.

Platelet-Derived Growth Factor-D Overexpression Contributes to Epithelial-Mesenchymal Transition of PC3 Prostate Cancer Cells

2008-06-12

The majority of human malignancies are believed to have epithelial origin, and the progression of cancer is often associated with a transient process named epithelial-mesenchymal transition (EMT). EMT is characterized by the loss of epithelial markers and the gain of mesenchymal markers that are typical of "cancer stem-like cells," which results in increased cell invasion and metastasis in vivo. Therefore, it is important to uncover the mechanistic role of factors that may induce EMT in cancer progression. Studies have shown that platelet-derived growth factor (PDGF) signaling contributes to EMT, and more recently, PDGF-D has been shown to regulate cancer cell invasion and angiogenesis. However, the mechanism by which PDGF-D promotes invasion and metastases and whether it is due to the acquisition of EMT phenotype remain elusive. For this study, we established stably transfected PC3 cells expressing high levels of PDGF-D, which resulted in the significant induction of EMT as shown by changes in cellular morphology concomitant with the loss of E-cadherin and zonula occludens-1 and gain of vimentin. We also found activation of mammalian target of rapamycin and nuclear factor-B, as well as Bcl-2 overexpression, in PDGF-D PC3 cells, which was associated with enhanced adhesive and invasive behaviors. More importantly, PDGF-D-overexpressing PC3 cells showed tumor growth in SCID mice much more rapidly than PC3 cells. These results provided a novel mechanism by which PDGF-D promotes EMT, which in turn increases tumor growth, and these results further suggest that PDGF-D could be a novel therapeutic target for the prevention and/or treatment of prostate cancer.

Disclosure of potential conflicts of interest is found at the end of this article.

Nucleofection Mediates High-Efficiency Stable Gene Knockdown and Transgene Expression in Human Embryonic Stem Cells

Hohenstein, K. A., Pyle, A. D., Chern, J. Y., Lock, L. F., Donovan, P. J. — 2008-06-12

High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells.

Disclosure of potential conflicts of interest is found at the end of this article.

Derivation and Cloning of a Novel Rhesus Embryonic Stem Cell Line Stably Expressing Tau-Green Fluorescent Protein

2008-06-12

Embryonic stem cells (ESC) have the ability of indefinite self-renewal and multilineage differentiation, and they carry great potential in cell-based therapies. The rhesus macaque is the most relevant preclinical model for assessing the benefit, safety, and efficacy of ESC-based transplantations in the treatment of neurodegenerative diseases. In the case of neural cell grafting, tracing both the neurons and their axonal projections in vivo is essential for studying the integration of the grafted cells in the host brain. Tau-Green fluorescent protein (tau-GFP) is a powerful viable lineage tracer, allowing visualization of cell bodies, dendrites, and axons in exquisite detail. Here, we report the first rhesus monkey ESC line that ubiquitously and stably expresses tau-GFP. First, we derived a new line of rhesus monkey ESC (LYON-ES1) that show marker expression and cell cycle characteristics typical of primate ESCs. LYON-ES1 cells are pluripotent, giving rise to derivatives of the three germ layers in vitro and in vivo through teratoma formation. They retain all their undifferentiated characteristics and a normal karyotype after prolonged culture. Using lentiviral infection, we then generated a monkey ESC line stably expressing tau-GFP that retains all the characteristics of the parental wild-type line and is clonogenic. We show that neural precursors derived from the tau-GFP ESC line are multipotent and that their fate can be precisely mapped in vivo after grafting in the adult rat brain.

Disclosure of potential conflicts of interest is found at the end of this article.

Selection Against Undifferentiated Human Embryonic Stem Cells by a Cytotoxic Antibody Recognizing Podocalyxin-Like Protein-1

2008-06-12

Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one antibody (mAb 84) is cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complement-independent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications.

Disclosure of potential conflicts of interest is found at the end of this article.

CXCR4+/FLK-1+ Biomarkers Select a Cardiopoietic Lineage from Embryonic Stem Cells

2008-06-12

Pluripotent stem cells demonstrate an inherent propensity for unrestricted multi-lineage differentiation. Translation into regenerative applications requires identification and isolation of tissue-specified progenitor cells. From a comprehensive pool of 11,272 quality-filtered genes, profiling embryonic stem cells at discrete stages of cardiopoiesis revealed 736 transcripts encoding membrane-associated proteins, where 306 were specifically upregulated with cardiogenic differentiation. Bioinformatic dissection of exposed surface biomarkers prioritized the chemokine receptor cluster as the most significantly over-represented gene receptor family during pre cardiac induction, with CXCR4 uniquely associated with mesendoderm formation. CXCR4⁺ progenitors were sorted from the embryonic stem cell pool into mesoderm-restricted progeny according to co-expression with the early mesoderm marker Flk-1. In contrast to CXCR4^–/Flk-1^– cells, the CXCR4⁺/Flk-1⁺ subpopulation demonstrated overexpressed cardiac lineage transcription factors (Mef2C, Myocardin, Nkx2.5), whereas pluripotent genes (Oct4, Fgf4, Sox2) as well as neuroectoderm (Sox1) and endoderm alpha-fetoprotein markers were all depleted. In fact, the CXCR4⁺/Flk-1⁺ biomarker combination identified embryonic stem cell progeny significantly enriched with Mesp-1, GATA-4, and Tbx5, indicative of pre cardiac mesoderm and the primary heart field. Although the CXCR4⁺/Flk-1⁺ transcriptome shared 97% identity with the CXCR4^–/Flk-1^– counterpart, the 818 divergent gene set represented predominantly cardiovascular developmental functions and formed a primitive cardiac network. Differentiation of CXCR4⁺/Flk-1⁺ progenitors yielded nuclear translocation of myocardial transcription factors and robust sarcomerogenesis with nascent cardiac tissue demonstrating beating activity and calcium transients. Thus, the CXCR4/Flk-1 biomarker pair predicts the emergence of cardiogenic specification within a pluripotent stem cell pool, enabling targeted selection of cardiopoietic lineage.

Disclosure of potential conflicts of interest is found at the end of this article.

Androgenetic Embryonic Stem Cells Form Neural Progenitor Cells In Vivo and In Vitro

2008-06-12

Uniparental zygotes with two paternal (androgenetic [AG]) or two maternal (gynogenetic [GG]; parthenogenetic [PG]) genomes are not able to develop into viable offspring but can form blastocysts from which embryonic stem cells (ESCs) can be derived. Although some aspects of the in vitro and in vivo differentiation potential of PG and GG ESCs of several species have been studied, the developmental capacity of AG ESCs is much less clear. Here, we investigate the potential of murine AG ESCs to undergo neural differentiation. We observed that AG ESCs differentiate in vitro into pan-neural progenitor cells (pnPCs) that further give rise to cells that express neuronal- and astroglial-specific markers. Neural progeny of in vitro-differentiated AG ESCs exhibited fidelity of expression of six imprinted genes analyzed, with the exception of Ube3a. Bisulfite sequencing for two imprinting control regions suggested that pnPCs predominantly maintained their methylation pattern. Following blastocyst injection of AG and biparental (normal fertilized [N]) ESCs, we found widespread and evenly distributed contribution of ESC-derived cells in both AG and N chimeric early fetal brains. AG and N ESC-derived cells isolated from chimeric fetal brains by fluorescence-activated cell sorting exhibited similar neurosphere-initiating cell frequencies and neural multilineage differentiation potential. Our results indicate that AG ESC-derived neural progenitor/stem cells do not differ from N neural progenitor/stem cells in their self-renewal and neural multilineage differentiation potential.

Disclosure of potential conflicts of interest is found at the end of this article.

Copy Number Variant Analysis of Human Embryonic Stem Cells

2008-06-12

Differences between individual DNA sequences provide the basis for human genetic variability. Forms of genetic variation include single-nucleotide polymorphisms, insertions/duplications, deletions, and inversions/translocations. The genome of human embryonic stem cells (hESCs) has been characterized mainly by karyotyping and comparative genomic hybridization (CGH), techniques whose relatively low resolution at 2–10 megabases (Mb) cannot accurately determine most copy number variability, which is estimated to involve 10%–20% of the genome. In this brief technical study, we examined HSF1 and HSF6 hESCs using array-comparative genomic hybridization (aCGH) to determine copy number variants (CNVs) as a higher-resolution method for characterizing hESCs. Our approach used five samples for each hESC line and showed four consistent CNVs for HSF1 and five consistent CNVs for HSF6. These consistent CNVs included amplifications and deletions that ranged in size from 20 kilobases to 1.48 megabases, involved seven different chromosomes, were both shared and unique between hESCs, and were maintained during neuronal stem/progenitor cell differentiation or drug selection. Thirty HSF1 and 40 HSF6 less consistently scored but still highly significant candidate CNVs were also identified. Overall, aCGH provides a promising approach for uniquely identifying hESCs and their derivatives and highlights a potential genomic source for distinct differentiation and functional potentials that lower-resolution karyotype and CGH techniques could miss.

Disclosure of potential conflicts of interest is found at the end of this article.

The Neuronal Differentiation Potential of Ldb1-Null Mutant Embryonic Stem Cells Is Dependent on Extrinsic Influences

Hwang, M., Gorivodsky, M., Kim, M., Westphal, H., Geum, D. — 2008-06-12

LIM-domain binding protein 1 (Ldb1) is a multiadaptor protein that mediates the action of transcription factors, including LIM-homeodomain proteins. To elucidate the functional role of Ldb1 in the neuronal differentiation of embryonic stem (ES) cells, we have generated Ldb1-null mutant (Ldb1–/–) ES cells and examined neuronal differentiation potentials in vitro using two different neuronal differentiation protocols. When subjected to a five-stage protocol that recapitulates in vivo conditions of neuronal differentiation, wild-type ES cells differentiated into a wide spectrum of neuronal cell types. However, Ldb1–/– ES cells did not differentiate into neuronal cells; instead, they differentiated into sarcomeric -actinin-positive muscle cells. In contrast, when an adherent monolayer culture procedure (which is based on the default mechanism of neural induction and eliminates environmental influences) was applied, both wild-type and Ldb1–/– ES cells differentiated into MAP2-positive mature neurons. Comparison of the results obtained when two different neuronal differentiation protocols were used suggests that Ldb1–/– ES cells have an innate potential to differentiate into neuronal cells, but this potential can be inhibited by environmental influences.

Disclosure of potential conflicts of interest is found at the end of this article.

Polycomb Repressive Complex 2 Is Dispensable for Maintenance of Embryonic Stem Cell Pluripotency

Chamberlain, S. J., Yee, D., Magnuson, T. — 2008-06-12

Polycomb repressive complex 2 (PRC2) methylates histone H3 tails at lysine 27 and is essential for embryonic development. The three core components of PRC2, Eed, Ezh2, and Suz12, are also highly expressed in embryonic stem (ES) cells, where they are postulated to repress developmental regulators and thereby prevent differentiation to maintain the pluripotent state. We performed gene expression and chimera analyses on low- and high-passage Eed^null ES cells to determine whether PRC2 is required for the maintenance of pluripotency. We report here that although developmental regulators are overexpressed in Eed^null ES cells, both low- and high-passage cells are functionally pluripotent. We hypothesize that they are pluripotent because they maintain expression of critical pluripotency factors. Given that EED is required for stability of EZH2, the catalytic subunit of the complex, these data suggest that PRC2 is not necessary for the maintenance of the pluripotent state in ES cells. We propose a positive-only model of embryonic stem cell maintenance, where positive regulation of pluripotency factors is sufficient to mediate stem cell pluripotency.

Disclosure of potential conflicts of interest is found at the end of this article.

Comprehensive MicroRNA Profiling Reveals a Unique Human Embryonic Stem Cell Signature Dominated by a Single Seed Sequence

2008-06-12

Embryonic stem cells are unique among cultured cells in their ability to self-renew and differentiate into a wide diversity of cell types, suggesting that a specific molecular control network underlies these features. Human embryonic stem cells (hESCs) are known to have distinct mRNA expression, global DNA methylation, and chromatin profiles, but the involvement of high-level regulators, such as microRNAs (miRNA), in the hESC-specific molecular network is poorly understood. We report that global miRNA expression profiling of hESCs and a variety of stem cell and differentiated cell types using a novel microarray platform revealed a unique set of miRNAs differentially regulated in hESCs, including numerous miRNAs not previously linked to hESCs. These hESC-associated miRNAs were more likely to be located in large genomic clusters, and less likely to be located in introns of coding genes. hESCs had higher expression of oncogenic miRNAs and lower expression of tumor suppressor miRNAs than the other cell types. Many miRNAs upregulated in hESCs share a common consensus seed sequence, suggesting that there is cooperative regulation of a critical set of target miRNAs. We propose that miRNAs are coordinately controlled in hESCs, and are key regulators of pluripotence and differentiation.

Disclosure of potential conflicts of interest is found at the end of this article.

Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells

Vazin, T., Chen, J., Lee, C.-T., Amable, R., Freed, W. J. — 2008-06-12

Producing dopaminergic (DA) neurons is a major goal of human embryonic stem cell (hESC) research. DA neurons can be differentiated from hESC by coculture with the mouse PA6 stromal cell line; this differentiation-inducing effect is termed stromal-derived inducing activity (SDIA). The molecular and biochemical nature of SDIA is, however, unknown. Various studies have suggested that SDIA involves either a fixation-resistant component located on the PA6 cell surface or factors secreted into the medium by PA6 cells. To address this question, hESC were cocultured with PA6 cells for 12 days and then further differentiated with sonic hedgehog homolog, fibroblast growth factor-8, and glial cell line-derived neurotrophic factor. After 18 days, 34% of cells were tyrosine hydroxylase (TH)+. When PA6 cells were fixed or irradiated, the number of TH+ cells was decreased by threefold, whereas mitomycin-c treatment of feeder cells decreased the number of TH+ cells by 32%. The neural-inducing effect of PA6 cells, as monitored by β-III-tubulin expression, was minimally affected by mitomycin-c treatment or fixation but was decreased 50% by irradiation. Medium conditioned by PA6 cells was ineffective in differentiating TH+ cells when used alone. Conditioned medium combined with heparin and/or fixed PA6 cells produced TH+ cell differentiation, although less effectively than PA6 cell coculture. Thus, PA6 cell surface activity is required for neural differentiation of hESC, but secreted factors are required for the specific DA neuron-inducing effect.

Disclosure of potential conflicts of interest is found at the end of this article.

Embryonic Stem Cell-Derived Pitx3-Enhanced Green Fluorescent Protein Midbrain Dopamine Neurons Survive Enrichment by Fluorescence-Activated Cell Sorting and Function in an Animal Model of Parkinson's Disease

2008-06-12

Both fetal ventral mesencephalic (VM) and embryonic stem (ES) cell-derived dopamine neurons have been used successfully to correct behavioral responses in animal models of Parkinson's disease. However, grafts derived from fetal VM cells or from ES cells contain multiple cell types, and the majority of these cells are not dopamine neurons. Isolation of ES cell-derived dopamine neurons and subsequent transplantation would both elucidate the capacity of these neurons to provide functional input and also further explore an efficient and safer use of ES cells for the treatment of Parkinson's disease. Toward this goal, we used a Pitx3-enhanced green fluorescent protein (Pitx3-eGFP) knock-in mouse blastocyst-derived embryonic stem (mES) cell line and fluorescence-activated cell sorting (FACS) to select and purify midbrain dopamine neurons. Initially, the dopaminergic marker profile of intact Pitx3-eGFP mES cultures was evaluated after differentiation in vitro. eGFP expression overlapped closely with that of Pitx3, Nurr1, Engrailed-1, Lmx1a, tyrosine hydroxylase (TH), l-aromatic amino acid decarboxylase (AADC), and vesicular monoamine transporter 2 (VMAT2), demonstrating that these cells were of a midbrain dopamine neuron character. Furthermore, postmitotic Pitx3-eGFP⁺ dopamine neurons, which constituted 2%–5% of all live cells in the culture after dissociation, could be highly enriched to >90% purity by FACS, and these isolated neurons were viable, extended neurites, and maintained a dopaminergic profile in vitro. Transplantation to 6-hydroxydopamine-lesioned rats showed that an enriched dopaminergic population could survive and restore both amphetamine- and apomorphine-induced functions, and the grafts contained large numbers of midbrain dopamine neurons, which innervated the host striatum.

Disclosure of potential conflicts of interest is found at the end of this article.

Reprogrammed Mouse Fibroblasts Differentiate into Cells of the Cardiovascular and Hematopoietic Lineages

2008-06-12

Forced expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the murine fibroblast genome, generating induced pluripotent stem (iPS) cells. Although the differentiation potential of these cells is thought to be equivalent to that of embryonic stem (ES) cells, it has not been rigorously determined. In this study, we sought to identify the capacity of iPS cells to differentiate into Flk1-positive progenitors and their mesodermal progeny, including cells of the cardiovascular and hematopoietic lineages. Immunostaining of tissues from iPS cell-derived chimeric mice demonstrated that iPS cells could contribute in vivo to cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. To compare the in vitro differentiation potential of murine ES and iPS cells, we either induced embryoid body (EB) formation of each cell type or cultured the cells on collagen type IV (ColIV), an extracellular matrix protein that had been reported to direct murine ES cell differentiation to mesodermal lineages. EB formation and exposure to ColIV both induced iPS cell differentiation into cells that expressed cardiovascular and hematopoietic markers. To determine whether ColIV-differentiated iPS cells contained a progenitor cell with cardiovascular and hematopoietic differentiation potential, Flk1-positive cells were isolated by magnetic cell sorting and exposed to specific differentiation conditions, which induced differentiation into functional cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. Our data demonstrate that murine iPS cells, like ES cells, can differentiate into cells of the cardiovascular and hematopoietic lineages and therefore may represent a valuable cell source for applications in regenerative medicine.

Disclosure of potential conflicts of interest is found at the end of this article.

Nuclear Magnetic Resonance Metabolomic Footprinting of Human Hepatic Stem Cells and Hepatoblasts Cultured in Hyaluronan-Matrix Hydrogels

2008-06-12

Human hepatoblasts (hHBs) and human hepatic stem cells (hHpSCs) were maintained in serum-free Kubota's medium, a defined medium tailored for hepatic progenitors, and on culture plastic versus hyaluronan hydrogels mixed with specific combinations of extracellular matrix components (e.g., type I collagen and laminin). Nuclear magnetic resonance spectroscopy was used to define metabolomic profiles for each substratum tested. The hHpSCs on culture plastic survived throughout the culture study, whereas hHBs on plastic died within 7–10 days. Both survived and expanded in all hydrogel-matrix combinations tested for more than 4 weeks. Profiles of hundreds of metabolites were narrowed to a detailed analysis of eight, such as glucose, lactate, and glutamine, shown to be significant components of cellular pathways, including the Krebs and urea cycles. The metabolomic profiles indicated that hHpSCs on plastic remained as stem cells expressing low levels of albumin but no -fetoprotein (AFP); those in hydrogels were primarily hHBs, expressing AFP, albumin, and urea. Both hHpSCs and hHBs used energy provided by anaerobic metabolism. Variations in hyaluronan-matrix chemistry resulted in distinct profiles correlating with growth or with differentiative responses. Metabolomic footprinting offers noninvasive and nondestructive assessment of physiological states of stem/progenitor cells ex vivo.

Disclosure of potential conflicts of interest is found at the end of this article.

Human Bone Marrow Adipocytes Block Granulopoiesis Through Neuropilin-1-Induced Granulocyte Colony-Stimulating Factor Inhibition

2008-06-12

Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34⁺ differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34⁺ cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflammatory cytokine such as interleukin-1 β or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes.

Disclosure of potential conflicts of interest is found at the end of this article.

Fibroblast Growth Factor 2 Maintains the Neurogenic Capacity of Embryonic Neural Progenitor Cells In Vitro but Changes Their Neuronal Subtype Specification

Bithell, A., Finch, S. E., Hornby, M. F., Williams, B. P. — 2008-06-12

Many in vitro systems used to examine multipotential neural progenitor cells (NPCs) rely on mitogens including fibroblast growth factor 2 (FGF2) for their continued expansion. However, FGF2 has also been shown to alter the expression of transcription factors (TFs) that determine cell fate. Here, we report that NPCs from the embryonic telencephalon grown without FGF2 retain many of their in vivo characteristics, making them a good model for investigating molecular mechanisms involved in cell fate specification and differentiation. However, exposure of cortical NPCs to FGF2 results in a profound change in the types of neurons generated, switching them from a glutamatergic to a GABAergic phenotype. This change closely correlates with the dramatic upregulation of TFs more characteristic of ventral telencephalic NPCs. In addition, exposure of cortical NPCs to FGF2 maintains their neurogenic potential in vitro, and NPCs spontaneously undergo differentiation following FGF2 withdrawal. These results highlight the importance of TFs in determining the types of neurons generated by NPCs in vitro. In addition, they show that FGF2, as well as acting as a mitogen, changes the developmental capabilities of NPCs. These findings have implications for the cell fate specification of in vitro-expanded NPCs and their ability to generate specific cell types for therapeutic applications.

Disclosure of potential conflicts of interest is found at the end of this article.

Neural Stem Cell Targeting of Glioma Is Dependent on Phosphoinositide 3-Kinase Signaling

2008-06-12

The utility of neural stem cells (NSCs) has extended beyond regenerative medicine to targeted gene delivery, as NSCs possess an inherent tropism to solid tumors, including invasive gliomas. However, for optimal clinical implementation, an understanding of the molecular events that regulate NSC tumor tropism is needed to ensure their safety and to maximize therapeutic efficacy. We show that human NSC lines responded to multiple tumor-derived growth factors and that hepatocyte growth factor (HGF) induced the strongest chemotactic response. Gliomatropism was critically dependent on c-Met signaling, as short hairpin RNA-mediated ablation of c-Met significantly attenuated the response. Furthermore, inhibition of Ras-phosphoinositide 3-kinase (PI3K) signaling impaired the migration of human neural stem cells (hNSCs) toward HGF and other growth factors. Migration toward tumor cells is a highly regulated process, in which multiple growth factor signals converge on Ras-PI3K, causing direct modification of the cytoskeleton. The signaling pathways that regulate hNSC migration are similar to those that promote unregulated glioma invasion, suggesting shared cellular mechanisms and responses.

Disclosure of potential conflicts of interest is found at the end of this article.

Sohlh2 Knockout Mice Are Male-Sterile Because of Degeneration of Differentiating Type A Spermatogonia

2008-06-12

The spermatogenesis and oogenesis-specific transcription factor Sohlh2 is normally expressed only in premeiotic germ cells. In this study, Sohlh2 and several other germ cell transcripts were found to be induced in mouse embryonic stem cells when cultured on a feeder cell line that overexpresses bone morphogenetic protein 4. To study the function of Sohlh2 in germ cells, we generated mice harboring null alleles of Sohlh2. Male Sohlh2-deficient mice were infertile because of a block in spermatogenesis. Although normal prior to birth, Sohlh2-null mice had reduced numbers of intermediate and type B spermatogonia by postnatal day 7. By day 10, development to the preleptotene spermatocyte stage was severely disrupted, rendering seminiferous tubules with only Sertoli cells, undifferentiated spermatogonia, and degenerating colonies of differentiating spermatogonia. Degenerating cells resembled type A2 spermatogonia and accumulated in M-phase prior to death. A similar phenotype was observed in Sohlh2-null mice on postnatal days 14, 21, 35, 49, 68, and 151. In adult Sohlh2-mutant mice, the ratio of undifferentiated type A spermatogonia (DAZL+/PLZF+) to differentiating type A spermatogonia (DAZL+/PLZF–) was twice normal levels. In culture, undifferentiated type A spermatogonia isolated from Sohlh2-null mice proliferated normally but linked the mutant phenotype to aberrant cell surface expression of the receptor-tyrosine kinase cKit. Thus, Sohlh2 is required for progression of differentiating type A spermatogonia into type B spermatogonia. One conclusion originating from these studies would be that testicular factors normally regulate the viability of differentiating spermatogonia by signaling through Sohlh2. This regulation would provide a crucial checkpoint to optimize the numbers of spermatocytes entering meiosis during each cycle of spermatogenesis.

Disclosure of potential conflicts of interest is found at the end of this article.

Autocrine Fibroblast Growth Factor 2 Increases the Multipotentiality of Human Adipose-Derived Mesenchymal Stem Cells

2008-06-12

Multipotent mesenchymal stem cells (MSCs), first identified in the bone marrow, have subsequently been found in many other tissues, including fat, cartilage, muscle, and bone. Adipose tissue has been identified as an alternative to bone marrow as a source for the isolation of MSCs, as it is neither limited in volume nor as invasive in the harvesting. This study compares the multipotentiality of bone marrow-derived mesenchymal stem cells (BMSCs) with that of adipose-derived mesenchymal stem cells (AMSCs) from 12 age- and sex-matched donors. Phenotypically, the cells are very similar, with only three surface markers, CD106, CD146, and HLA-ABC, differentially expressed in the BMSCs. Although colony-forming units-fibroblastic numbers in BMSCs were higher than in AMSCs, the expression of multiple stem cell-related genes, like that of fibroblast growth factor 2 (FGF2), the Wnt pathway effectors FRAT1 and frizzled 1, and other self-renewal markers, was greater in AMSCs. Furthermore, AMSCs displayed enhanced osteogenic and adipogenic potential, whereas BMSCs formed chondrocytes more readily than AMSCs. However, by removing the effects of proliferation from the experiment, AMSCs no longer out-performed BMSCs in their ability to undergo osteogenic and adipogenic differentiation. Inhibition of the FGF2/fibroblast growth factor receptor 1 signaling pathway demonstrated that FGF2 is required for the proliferation of both AMSCs and BMSCs, yet blocking FGF2 signaling had no direct effect on osteogenic differentiation.

Disclosure of potential conflicts of interest is found at the end of this article.

A Putative Role for RHAMM/HMMR as a Negative Marker of Stem Cell-Containing Population of Human Limbal Epithelial Cells

2008-06-12

The corneal epithelium is maintained by stem cells located at the periphery of the cornea in a region known as the limbus. Depletion of limbal stem cells (LSCs) results in limbal stem cell deficiency. Treatments for this disease are based on limbal replacement or transplantation of ex vivo expanded LSCs. It is, therefore, crucial to identify cell surface markers for LSCs that can be used for their enrichment and characterization. Aldehyde dehydrogenases (ALDHs) are enzymes which protect cells from the toxic effects of peroxidic aldehydes. In this manuscript, we show for the first time that ALDH1 is absent from the basal cells of the limbal and corneal epithelium. We separated limbal epithelial cells on the basis of ALDH activity and showed that ALDH^dim cells expressed significantly higher levels of Np63 and ABCG2 as well as having a greater colony forming efficiency (CFE) when compared to ALDH^bright cells. Large scale transcriptional analysis of these two populations led to identification of a new cell surface marker, RHAMM/HMMR, which is located in all layers of corneal epithelium and in the suprabasal layers of the limbal epithelium but is completely absent from the basal layer of the limbus. Our studies indicate that absence of RHAMM/HMMR expression is correlated with properties associated with LSCs. RHAMM/HMMR- limbal epithelial cells are smaller in size, express negligible CK3, have higher levels of Np63 and have a higher CFE compared to RHAMM/HMMR+ cells. Taken together these results suggest a putative role for RHAMM/ HMMR as a negative marker of stem cell containing limbal epithelial cells. Cell selection based on Hoechst exclusion and lack of cell surface RHAMM/HMMR expression resulted in increased colony forming efficiency compared to negative selection using RHAMM/HMMR alone or positive selection using Hoechst on its own. Combination of these two cell selection methods presents a novel method for LSC enrichment and characterization.

Disclosure of potential conflicts of interest is found at the end of this article.

Sustained Long-Term Engraftment and Transgene Expression of Peripheral Blood CD34+ Cells Transduced with Third-Generation Lentiviral Vectors

2008-06-12

As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34⁺ cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34⁺ cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34⁺ (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB.

Disclosure of potential conflicts of interest is found at the end of this article.

Insulin-Like Growth Factor-Binding Protein 2 Secreted by a Tumorigenic Cell Line Supports Ex Vivo Expansion of Mouse Hematopoietic Stem Cells

2008-06-12

Successful hematopoietic stem cell (HSC) transplantation is often limited by the numbers of HSCs, and robust methods to expand HSCs ex vivo are needed. We previously showed that angiopoietin-like proteins (Angptls), a group of growth factors isolated from a fetal liver HSC-supportive cell population, improved ex vivo expansion of HSCs. Here, we demonstrate that insulin-like growth factor-binding protein 2 (IGFBP2), secreted by a tumorigenic cell line, also enhanced ex vivo expansion of mouse HSCs. On the basis of these findings, we established a completely defined, serum-free culture system for mouse HSCs, containing SCF, thrombopoietin, fibroblast growth factor 1, Angptl3, and IGFBP2. As measured by competitive repopulation analyses, there was a 48-fold increase in numbers of long-term repopulating mouse HSCs after 21 days of culture. This is the first demonstration that IGFBP2 stimulates expansion or proliferation of murine stem cells. Our finding also suggests that certain cancer cells synthesize proteins that can stimulate HSC expansion.

Disclosure of potential conflicts of interest is found at the end of this article.

Neuropeptide Y Promotes Neurogenesis in Murine Subventricular Zone

2008-06-12

Stem cells of the subventricular zone (SVZ) represent a reliable source of neurons for cell replacement. Neuropeptide Y (NPY) promotes neurogenesis in the hippocampal subgranular layer and the olfactory epithelium and may be useful for the stimulation of SVZ dynamic in brain repair purposes. We describe that NPY promotes SVZ neurogenesis. NPY (1 µM) treatments increased proliferation at 48 hours and neuronal differentiation at 7 days in SVZ cell cultures. NPY proneurogenic properties are mediated via the Y1 receptor. Accordingly, Y1 receptor is a major active NPY receptor in the mouse SVZ, as shown by functional autoradiography. Moreover, short exposure to NPY increased immunoreactivity for the phosphorylated form of extracellular signal-regulated kinase 1/2 in the nucleus, compatible with a trigger for proliferation, whereas 6 hours of treatment amplified the phosphorylated form of c-Jun-NH₂-terminal kinase signal in growing axons, consistent with axonogenesis. NPY, as a promoter of SVZ neurogenesis, is a crucial factor for future development of cell-based brain therapy.

Disclosure of potential conflicts of interest is found at the end of this article.

Transplantation of Bone Marrow-Derived Very Small Embryonic-Like Stem Cells Attenuates Left Ventricular Dysfunction and Remodeling After Myocardial Infarction

2008-06-12

Adult bone marrow (BM) contains Sca-1+/Lin–/CD45– very small embryonic-like stem cells (VSELs) that express markers of several lineages, including cardiac markers, and differentiate into cardiomyocytes in vitro. We examined whether BM-derived VSELs promote myocardial repair after a reperfused myocardial infarction (MI). Mice underwent a 30-minute coronary occlusion followed by reperfusion and received intramyocardial injection of vehicle (n= 11), 1 x 10⁵ Sca-1+/Lin–/CD45+ enhanced green fluorescent protein (EGFP)-labeled hematopoietic stem cells (n= 13 [cell control group]), or 1 x 10⁴ Sca-1+/Lin–/CD45– EGFP-labeled cells (n= 14 [VSEL-treated group]) at 48 hours after MI. At 35 days after MI, VSEL-treated mice exhibited improved global and regional left ventricular (LV) systolic function (echocardiography) and attenuated myocyte hypertrophy in surviving tissue (histology and echocardiography) compared with vehicle-treated controls. In contrast, transplantation of Sca-1+/Lin–/CD45+ cells failed to confer any functional or structural benefits. Scattered EGFP+ myocytes and capillaries were present in the infarct region in VSEL-treated mice, but their numbers were very small. These results indicate that transplantation of a relatively small number of CD45– VSELs is sufficient to improve LV function and alleviate myocyte hypertrophy after MI, supporting the potential therapeutic utility of these cells for cardiac repair.

Disclosure of potential conflicts of interest is found at the end of this article.

Totipotency/Pluripotency and Patentability

Denker, H.-W. — 2008-06-12

In their article entitled "Commentary: Is totipotency of a human cell a sufficient reason to exclude its patentability under the European law" (Stem Cells 2007;25:3026–3028), K.T. Vrtovec and B. Vrtovec conclude that arguments based on differentiation potential should not be an obstacle to patenting human embryonic stem cells (and related cells referred to as totipotent or pluripotent). While concentrating on formal legal aspects, however, these authors fail to consider a major biological and ethical argument already found in the literature, namely that an obstacle to patenting is to be seen in the potential of cells (e.g., of embryonic stem cell lines), if this potential allows (re)constitution of an embryo when tetraploid complementation is performed.

Disclosure of potential conflicts of interest is found at the end of this article.

Impairment of Granulo-Monocytic Development of Human Common Myeloid Progenitors but Not of Granulo-Monocytic Progenitors by Decreasing Stem Cell Leukemia/T-Cell Acute Leukemia 1 Expression

Brunet de la Grange, P., Zink, E., Armstrong, F., Rouyez, M.-C., Pflumio, F. — 2008-06-12

We recently showed that Stem Cell Leukemia/T-cell Acute Leukemia 1 (SCL/TAL1) regulates hematopoiesis from hematopoietic stem cells to committed myeloid progenitors compartment. However, in this heterogeneous compartment, the precise role of TAL1, that is largely debated, remains to be clearly defined, notably at the common myeloid progenitor (CMP) and granulo-monocytic progenitor (GMP) levels. Using small hairpin (sh)RNA lentiviral constructs, we decreased TAL1 expression in sorted human CMP and GMP subpopulations that were then assayed for erythroid and granulo-monocytic (GM) differentiation. Decreased TAL1 expression in CMP resulted in rare erythroid colonies, in a 2–3 fold reduction of GM colony number in clonogenic assays and in a 3.6–5.6 decreased production of CD14⁺CD15⁺ GM cells in liquid culture. Moreover, analysis of transcript profile of gene involved in GM differentiation showed that GM cells expressing shRNA-TAL1 construct displayed decreased levels of g-csfr, c/ebp, and mpo and high levels of gata-2 transcripts, indicating a blocking of GM differentiation. In contrast, GM differentiation of GMP remained unaffected when TAL1 transcript levels were decreased. These data definitively delineate the human myeloid progenitors that are regulated by TAL1.

Disclosure of potential conflicts of interest is found at the end of this article.