Pluripotent Stem Cells for Modelling and Cell Therapy of Parkinson’s Disease

Studying pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD), requires adequate disease models. The available patient’s material is limited to biological fluids and post mortem brain samples. Disease modeling and drug screening can be done in animal models, although this approach has its own limitations, since laboratory animals do not suffer from many neurodegenerative diseases, including PD. The use of neurons obtained by targeted differentiation from induced pluripotent stem cells (iPSCs) with known genetic mutations, as well as from carriers of sporadic forms of the disease, will allow to elucidate new components of the molecular mechanisms of neurodegeneration. Such neuronal cultures can also serve as unique models for testing neuroprotective compounds and monitoring neurodegenerative changes against a background of various therapeutic interventions. In the future, dopaminergic neurons differentiated from iPSCs can be used for cell therapy of PD.     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Proliferating Patent Problems with Human Embryonic Stem Cell Research?

The scientific challenges and ethical controversies facing human embryonic stem cell (hESC) research continue to command attention. The issues posed by patenting hESC technologies have, however, largely failed to penetrate the discourse, much less result in political action. This paper examines U.S. and European patent systems, illustrating discrepancies in the patentability of hESC technologies and identifying potential negative consequences associated with efforts to make available hESC research tools for basic research purposes while at same time strengthening the position of certain patent-holders' rights. Differences between the U.S. and the European contexts may in part explain why the course of hESC research in those jurisdictions ultimately diverges. Nevertheless, questions about whether and how patenting, related agreements, and licensing practices progress and shape the field of hESC research in both the U.S., Europe, and elsewhere must no longer be marginalised. These questions are fundamentally important in determining what benefits are likely to result from hESC research. Assuring these benefits is the moral issue with which patent systems are most intrinsically concerned, and that governments must begin to directly address rather than assume or ignore.     

Defined Essential 8? Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm² of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x10⁶ cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.     

Generation of Human Induced Pluripotent Stem (iPS) Cells in Serum- and Feeder-Free Defined Culture and TGF-β1 Regulation of Pluripotency

Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka''s factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-β1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-β1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-β1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.     

Mass Spectrometry–based Proteomic Analysis of the Matrix Microenvironment in Pluripotent Stem Cell Culture

The cellular microenvironment comprises soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to regulate cellular behavior. Pluripotent stem cells utilize interactions between support cells and soluble factors in the microenvironment to assist in the maintenance of self-renewal and the process of differentiation. However, the ECM also plays a significant role in shaping the behavior of human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells. Moreover, it has recently been observed that deposited factors in a hESC-conditioned matrix have the potential to contribute to the reprogramming of metastatic melanoma cells. Therefore, the ECM component of the pluripotent stem cell microenvironment necessitates further analysis.In this study we first compared the self-renewal and differentiation properties of hESCs grown on Matrigel™ pre-conditioned by hESCs to those on unconditioned Matrigel™. We determined that culture on conditioned Matrigel™ prevents differentiation when supportive growth factors are removed from the culture medium. To investigate and identify factors potentially responsible for this beneficial effect, we performed a defined SILAC MS-based proteomics screen of hESC-conditioned Matrigel™. From this proteomics screen, we identified over 80 extracellular proteins in matrix conditioned by hESCs and induced pluripotent stem cells. These included matrix-associated factors that participate in key stem cell pluripotency regulatory pathways, such as Nodal/Activin and canonical Wnt signaling. This work represents the first investigation of stem-cell-derived matrices from human pluripotent stem cells using a defined SILAC MS-based proteomics approach.The two defining characteristics of human embryonic stem cells (hESCs),¹ self-renewal and pluripotency, are maintained by a delicate balance of intracellular and extracellular signaling processes. Extracellular regulation is primarily the result of changes in the microenvironment surrounding the cells during growth in vitro or in vivo. HESCs interact with this “niche ” through support cells, extracellular matrix (ECM) components, and autocrine/paracrine signaling (reviewed in Refs. 1–3). Modulation of any of these supportive elements individually or in combination has been used extensively to alter hESC behavior (1–3).The culture of hESCs, as well as that of human induced pluripotent stem cells (hiPSCs), is conventionally performed on a layer of irradiated mouse embryonic fibroblast cells (MEFs). These MEFs are believed to promote the maintenance of hESCs and hiPSCs through the secretion of beneficial support proteins and cytokines into the soluble microenvironment. A number of proteomic studies have been conducted that examine the secretome of feeder-cell layers in an attempt to elucidate proteins and pathways essential for hESC and hiPSC survival (4–7). Alternatively, hESCs and hiPSCs can be cultured in feeder-free conditions in the absence of support cells. In feeder-free conditions, hESCs and hiPSCs are most often grown on the basement membrane matrix Matrigel™ in medium that has been previously conditioned by MEFs (MEF-CM). Matrigel™ is a gelatinous mixture that is secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (8). Although recent studies have proposed that a variety of defined matrices can support the growth of hESCs and hiPSCs, few of these can maintain a wide range of stem cell lines and therefore are typically not used in place of Matrigel™. The properties of Matrigel™ that make it such an effective matrix for hESC and hiPSC culture remain poorly understood. Because of the complexity of matrices like Matrigel™, the majority of proteomic studies that examine the hESC and hiPSC microenvironment have focused on contributions from support cells and soluble extracellular factors.The ECM is typically a complex network of structural proteins and glycosaminoglycans that function to support cells through the regulation of processes such as adhesion and growth factor signaling (9). Thus, it is not surprising that the generation of a well-defined matrix capable of facilitating hESC and hiPSC self-renewal has remained difficult (10). Previous proteomic investigations of Matrigel™ and other matrices supportive of hESC maintenance in vitro have revealed the presence of numerous growth, binding, and signaling proteins (11, 12). Further examination of how hESCs and hiPSCs interact with these complex matrices would provide critical information about what role the ECM plays in the organization of processes involved in the regulation of self-renewal and pluripotency.A recent study has established the ability of hESC-derived matrix microenvironments to alter tumorigenic properties through the reprogramming of metastatic melanoma cells (13). Importantly, this effect was found to be dependent on the exposure of metastatic cells to hESC-derived conditioned Matrigel™. Culture of metastatic melanoma cells in hESC-conditioned medium did not promote the reprogramming effect. These data suggest that the proteins responsible for this effect were integrated in the matrix. With the use of immunochemical techniques, it was later found that the left-right determination (Lefty) proteins A and B that were deposited in the matrix by hESCs during conditioning were at least in part responsible for the cellular change observed in metastatic cells (14). The Lefty A and B proteins are antagonists of transforming growth factor (TGF)-β signaling that act directly on Nodal protein, a critical regulator of the stem cell phenotype (15, 16). Subsequent studies of conditioned matrix utilizing mESCs implicated the bone morphogenic protein (BMP) 4 antagonist Gremlin as a primary regulator of the observed changes in metastatic cells (17). Collectively, these studies were all biased by a targeted analysis of potential effectors of metastatic cells. A comprehensive proteomic analysis of conditioned matrix could potentially reveal other factors involved in metastatic cell reprogramming. Furthermore, proteomic examination of hESC and hiPSC conditioned matrix could expose factors important in the regulation of self-renewal and pluripotency by the microenvironment in vitro.To this end, we have analyzed both types of human pluripotent stem cells, hESCs and hiPSCs, via a mass spectrometry (MS)-based proteomics approach to identify proteins deposited during growth in feeder-free conditions in vitro on Matrigel™. To investigate the hESC- and hiPSC-derived matrix, the metabolic labeling technique known as stable isotope labeling with amino acids in cell culture (SILAC) was used (18). SILAC facilitates the identification of hESC- and hiPSC-derived proteins that would otherwise be confounded by the presence of mouse-derived protein background from Matrigel™. From the proteomic analysis of three cells lines, namely, the hESC lines H9 and CA1 and the hiPSC line BJ-1D, we identified a total of 621, 1355, and 1350 total unique proteins, respectively. This work represents the first analysis of a hESC- and hiPSC-derived conditioned matrix and resulted in the identification of at least one novel microenvironmental contributor responsible for the regulation of human pluripotent stem cells.     

Suppressing NF-κB and NKRF Pathways by Induced Pluripotent Stem Cell Therapy in Mice with Ventilator-Induced Lung Injury

Background

High-tidal-volume mechanical ventilation used in patients with acute lung injury (ALI) can induce the release of inflammatory cytokines, as macrophage inflammatory protein-2 (MIP-2), recruitment of neutrophils, and disruption of alveolar epithelial and endothelial barriers. Induced pluripotent stem cells (iPSCs) have been shown to improve ALI in mice, but the mechanisms regulating the interactions between mechanical ventilation and iPSCs are not fully elucidated. Nuclear factor kappa B (NF-κB) and NF-κB repressing factor (NKRF) have been proposed to modulate the neutrophil activation involved in ALI. Thus, we hypothesized intravenous injection of iPSCs or iPSC-derived conditioned medium (iPSC-CM) would decrease high-tidal-volume ventilation-induced neutrophil infiltration, oxidative stress, and MIP-2 production through NF-κB/NKRF pathways.

Methods

Male C57BL/6 mice, aged between 6 and 8 weeks, weighing between 20 and 25 g, were exposed to high-tidal-volume (30 ml/kg) mechanical ventilation with room air for 1 to 4 h after 5×10⁷ cells/kg mouse iPSCs or iPSC-CM administration. Nonventilated mice were used as control groups.

Results

High-tidal-volume mechanical ventilation induced the increases of integration of iPSCs into the injured lungs of mice, microvascular permeability, neutrophil infiltration, malondialdehyde, MIP-2 production, and NF-κB and NKRF activation. Lung injury indices including inflammation, lung edema, ultrastructure pathologic changes and functional gas exchange impairment induced by mechanical ventilation were attenuated with administration of iPSCs or iPSC-CM, which was mimicked by pharmacological inhibition of NF-κB activity with SN50 or NKRF expression with NKRF short interfering RNA.

Conclusions

Our data suggest that iPSC-based therapy attenuates high-tidal-volume mechanical ventilation-induced lung injury, at least partly, through inhibition of NF-κB/NKRF pathways. Notably, the conditioned medium of iPSCs revealed beneficial effects equal to those of iPSCs.     

Regulation of Induced Pluripotent Stem (iPS) Cell Induction by Wnt/β-Catenin Signaling

Wnt signaling has been implicated in promoting somatic cell reprogramming. However, its molecular mechanisms remain unknown. Here we report that Wnt/β-catenin enhances iPSCs induction at the early stage of reprogramming. The augmented reprogramming induced by β-catenin is not due to increased total cell population or activation of c-Myc. In addition, β-catenin interacts with reprogramming factors Klf4, Oct4, and Sox2, further enhancing expression of pluripotency circuitry genes. These studies reveal novel mechanisms underlying the regulation of reprogramming somatic cells to pluripotency by Wnt/β-catenin signaling.     

Cancer: Inappropriate Expression of Stem Cell Programs?

Cancer stem cells (CSCs) are a subpopulation of cancer cells that possess characteristics, including self-renewal, associated with normal stem cells. In this issue of Cell Stem Cell, Wong et al. (2008) define a core embryonic stem cell (ESC)-like gene expression program that may be important for CSC function in multiple epithelial cancers.     

Effect of Human WEE1 and Stem Cell Factor on Human CD34^＋ Umbilical Cord Blood Cell Damage Induced by Chemotherapeutic Agents

Myelosuppression is one of the major side-effects of most anticancer drugs. To achieve myeloprotection, one bicistronic vector encoding anti-apoptotic protein human WEE l （WEElHu） and proliferation-stimulating stem cell factor （SCF） was generated. In this study, we selected human umbilical cord blood CD34^＋ cells as the in vitro model in an attempt to investigate whether WEEIHu, rather than conventional drug-resistant genes, can be introduced to rescue cells from the damage by chemotherapeutic agents such as cisplatin, adriamycin, mitomycin-c and 5-fluorouracil. Cell viability and cytotoxicity assay, colony-forming units in culture assay and externalization of phospholipid phosphatidylserine analysis showed that the expression of WEElHu and SCF in CD34^＋ cells provided the cells with some protection. These findings suggest that the expression of WEElHu and SCF might rescue CD34^＋ cells from chemotherapyinduced myelosuppression.     

At Home among Strangers: Is It Possible to Create Hypoimmunogenic Pluripotent Stem Cell Lines?

Molecular Biology - Human pluripotent stem cells, which include embryonic stem cells and induced pluripotent cells (iPSCs), are capable of unlimited division and differentiation into all cells of...     

Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.     

Genomic Instability Associated with p53 Knockdown in the Generation of Huntington’s Disease Human Induced Pluripotent Stem Cells

Alterations in DNA damage response and repair have been observed in Huntington’s disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown.