Francesca S. Boscolo

Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells

Our understanding of Alzheimer’s disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer’s disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APPDp), two with sporadic Alzheimer’s disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APPDp patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1–40), phospho-tau(Thr 231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APPDp and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer’s disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer’s disease, even though it can take decades for overt disease to manifest in patients.

Recurrent Variations in DNA Methylation in Human Pluripotent Stem Cells and Their Differentiated Derivatives

Human pluripotent stem cells (hPSCs) are potential sources of cells for modeling disease and development, drug discovery, and regenerative medicine. However, it is important to identify factors that may impact the utility of hPSCs for these applications. In an unbiased analysis of 205 hPSC and 130 somatic samples, we identified hPSC-specific epigenetic and transcriptional aberrations in genes subject to X chromosome inactivation (XCI) and genomic imprinting, which were not corrected during directed differentiation. We also found that specific tissue types were distinguished by unique patterns of DNA hypomethylation, which were recapitulated by DNA demethylation during in vitro directed differentiation. Our results suggest that verification of baseline epigenetic status is critical for hPSC-based disease models in which the observed phenotype depends on proper XCI or imprinting and that tissue-specific DNA methylation patterns can be accurately modeled during directed differentiation of hPSCs, even in the presence of variations in XCI or imprinting.

Conversion of human fibroblasts to angioblast-like progenitor cells

Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies.

Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.

Deriving dopaminergic neurons for clinical use. A practical approach

New small molecules that regulate the step-wise differentiation of human pluripotent stem cells into dopaminergic neurons have been identified. The steroid, guggulsterone, was found to be the most effective inducer of neural stem cells into dopaminergic neurons. These neurons are extensively characterized and shown to be functional. We believe this new approach offers a practical route to creating neurons of sufficient quality to be used to treat Parkinsons disease patients.

Melanocytes Derived from Transgene-Free Human Induced Pluripotent Stem Cells

TO THE EDITOR Defects in melanocytes have been implicated in the etiology of a variety of human skin diseases and disorders (Lin and Fisher, 2007; Fistarol and Itin, 2010; Rees, 2011). There is long-standing interest in studying the development and dysfunction of human melanocytes, but there has not been a reliable and accessible system to study early events in human melanocyte differentiation. An in vitro system that reliably and efficiently produces normal human melanocytes from embryonic stage cells would allow us to better dissect the physiological and pathological development of melanocytes. Recent advances in stem cell biology have led to the establishment of human induced pluripotent stem cell (hiPSC) techniques that enable researchers to reprogram somatic cells to the pluripotent state (Takahashi et al., 2007). Differentiation of human and mouse pluripotent stem cells (PSCs) toward the melanocyte lineage has been reported (Yamane et al., 1999; Pla et al., 2005; Fang et al., 2006; Nissan et al., 2011; Ohta et al., 2011; Yang et al., 2011), but existing protocols have shortcomings that may limit their research and clinical applications. For example, the use of embryonic stem cells could lead to allogeneic immunoincompatibility of differentiated melanocytes and transplant recipients. In addition, the use of hiPSCs generated by integrative reprogramming strategies raises concerns about reactivation of retained transgenes, some of which are oncogenes. In addition, the current methods for melanocyte differentiation from hiPSCs require optimization in order to reproducibly generate high-purity melanocytes from multiple hiPSC lines. We have established a strategy to produce human melanocytes in vitro for use as a platform for pigment cell research and the development of cell-based therapies. We first derived transgene-free hiPSCs from two distinct types of skin cells: human primary melanocytes (HMs) and human dermal fibroblasts (HDF51) (Figure 1a and Supplementary Figure S1a online). We used a nonintegrative reprogramming approach mediated by Sendai virus–based vectors independently encoding POU5F1, SOX2, KLF4, and MYC (Fusaki et al., 2009; Macarthur et al., 2012). As shown in Figure 1b and Supplementary Figure S1b online, biomarkers of cellular pluripotency, including endogenous OCT4/POU5F1, NANOG, Tra-1-81, and UEA-I (Wang et al., 2011), were positive in HMi-506, HMi-503, and HDF51i-509 hiPSCs. Cells were also shown to be pluripotent using a gene expression diagnostic test (PluriTest; Muller et al., 2011), by differentiation into cells that express biomarkers relevant to all three germ layers in vitro (Figure 1c and Supplementary Figure S1c, S1d and S1e online) and by generation of teratomas (Supplementary Figure S1d online). Figure 1 Generation and differentiation of transgene-free human induced pluripotent stem cell (hiPSCs). (a) HMi-506 cells generated from human primary melanocyte (HM) cells using a Sendai virus–based reprogramming system were cocultured with mouse embryonic ... We newly developed two differentiation protocols based on previously reported methods. One protocol involves an aggregation-in-suspension step, whereas the other does not (Supplementary Figure S2 online). Both protocols generated cells displaying typical melanocyte morphology and pigmentation (Figure 1d) from hiPSCs after 30 days of directed differentiation, suggesting that the aggregation-in-suspension step is dispensable. The melanin granules that accumulated at the dendritic tips of differentiated cells were intensely stained by Fontana–Masson staining, indicating that the pigmentation of these cells was due to melanogenesis (Supplementary Figure S3 online). In addition, MITF (microphthalmia-associated transcription factor), a marker for melanocyte progenitors, was expressed in more than 90% of the differentiated derivatives after 30 days (Figure 1e and Supplementary Figure S4 online), which appears to be a higher differentiation efficiency than other reported protocols (Nissan et al., 2011; Ohta et al., 2011). As expected, MITF was not detected in the undifferentiated hiPSCs, and was present in the primary melanocytes (Figure 1e). Notably, our protocols resulted in similarly high levels of melanocyte differentiation for all four independent hiPSC lines examined, highlighting their reproducibility. Other melanocytic biomarkers including TYR (tyrosinase), MLANA (melan-A), TYRP1 (tyrosinase-related protein 1), PMEL (premelanosome protein), PAX3 (paired box 3), and SOX10 (SRY-box 10) were highly expressed in the differentiated derivatives (similar to primary melanocytes, Figure 2a and b). The melanin content and cell signaling involved in melanin production in the differentiated derivatives was increased by treatment with α-melanocyte-stimulating hormone (α-MSH) in a dose-dependent manner (Figure 2c and d and Supplementary Figure S5 online). These findings indicate that the differentiated derivatives possess molecular features of bona fide melanocytes and accurately mimic their ability to respond to α-MSH, which is the factor that activates melanogenesis and enhances skin pigmentation during the tanning response (Thody, 1999). Figure 2 Molecular and functional characterization of the melanocyte-like differentiated cells. (a) Heat map and dendrogram of melanocytic biomarkers showing that these transcripts were preferentially expressed in human primary melanocyte (HM) cells and HMi-506_Mel ... Genome-wide gene expression profiling and unsupervised hierarchical clustering revealed that the melanocytes (HMi-506_Mel Diff_1 and HMi-506_Mel Diff_2) differentiated from the HMi-506 cells were closely clustered with HMs and were distinct from all undifferentiated hiPSC samples (Figure 2e). As genetic abnormalities may occur in hiPSC genomes during the reprogramming and differentiation processes, we tested the genomic stability of the cells by comparing the differentiated derivatives with the parental primary melanocytes using high-resolution single-nucleotide polymorphism (SNP) genotyping and copy number variation analysis. As shown in Figure 2f, the HMi-506_Mel Diff derivatives and parental cells showed highly similar genotyping profiles, showing that the cellular genome remained stable during reprogramming and differentiation. Similar to human melanocytes in vivo, the differentiated derivatives in semiautologous skin reconstructs were located at the dermis–epidermis interface and interspersed with keratinocytes (Supplementary Figure S6a, S6b, S6c and S6d online), indicating their ability to integrate with the skin tissue of transplant recipients. Similar to the autologous dermal fibroblasts used for generating transgene-free hiPSCs, the differentiated derivatives stimulated limited proliferation of peripheral blood mononuclear cells that were isolated from the blood of the same individual in a mixed lymphocyte reaction assay (Supplementary Figure S6e online). These results attest to the clinical advantages of melanocytes differentiated from hiPSCs using the reprogramming and differentiation approaches described here. In this study, we have demonstrated that genetically stable melanocytes can be efficiently differentiated from transgene-free hiPSCs generated from two different types of cutaneous cells. This differentiation protocol takes less time than previously reported melanocytic differentiation protocols, and we showed that it is equally effective for multiple independent hiPSC lines. We performed a thorough investigation of the differentiated cells, including genome-wide gene expression analysis and SNP genotyping in addition to functional assays. Our approach can serve as an unlimited source of custom human melanocytes that can be used for novel approaches for modeling human skin disease (e.g., melanoma and vitiligo) and to provide material for transplantation.

Banking placental tissue: An optimized collection procedure for genome-wide analysis of nucleic acids

INTRODUCTION Banking of high-quality placental tissue specimens will enable biomarker discovery and molecular studies on diseases involving placental dysfunction. Systematic studies aimed at developing feasible standardized methodology for placental collection in a typical clinical setting are lacking. METHODS To determine the acceptable timeframe for placental collection, we collected multiple samples from first and third trimester placentas at serial timepoints in a 2-h window after delivery, simultaneously comparing the traditional snap-freeze technique to commercial solutions designed to preserve RNA (RNAlater™), and DNA (DNAgard(®)). The performance of RNAlater for preserving DNA was also tested. Nucleic acid quality was assessed by determining the RNA integrity number (RIN) and genome-wide microarray profiling for gene expression and DNA methylation. RESULTS We found that samples collected in RNAlater had higher and more consistent RINs compared to snap-frozen tissue. Similar RINs were obtained for tissue collected in RNAlater as large (1 cm(3)) and small (∼0.1 cm(3)) pieces. RNAlater appeared to better stabilize the time zero gene expression profile compared to snap-freezing for first trimester placenta. DNA methylation profiles remained quite stable over a 2 h time period after removal of the placenta from the uterus, with DNAgard being superior to other treatments. DISCUSSION AND CONCLUSION The collection of placental samples in RNAlater and DNAgard is simple, and eliminates the need for liquid nitrogen or a freezer on-site. Moreover, the quality of the nucleic acids and the resulting data from samples collected in these preservation solutions is higher than samples collected using the snap-freeze method and easier to implement in busy clinical environments.

Genomic Analysis of hESC Pedigrees Identifies De Novo Mutations and Enables Determination of the Timing and Origin of Mutational Events

Summary Given the association between mutational load and cancer, the observation that genetic aberrations are frequently found in human pluripotent stem cells (hPSCs) is of concern. Prior studies in human induced pluripotent stem cells (hiPSCs) have shown that deletions and regions of loss of heterozygosity (LOH) tend to arise during reprogramming and early culture, whereas duplications more frequently occur during long-term culture. For the corresponding experiments in human embryonic stem cells (hESCs), we studied two sets of hESC lines: one including the corresponding parental DNA and the other generated from single blastomeres from four sibling embryos. Here, we show that genetic aberrations observed in hESCs can originate during preimplantation embryo development and/or early derivation. These early aberrations are mainly deletions and LOH, whereas aberrations arising during long-term culture of hESCs are more frequently duplications. Our results highlight the importance of close monitoring of genomic integrity and the development of improved methods for derivation and culture of hPSCs.

Culture of Human Pluripotent Stem Cells in Feeder-Free Conditions

There are multiple methods for culturing hPSCs under feeder-free conditions. Here we describe a system using StemPro medium and Geltrex or Matrigel substrata with Accutase passaging. In our hands hPSCs cultured this way remain pluripotent and karyotypically normal for more than 40 passages. The advantage of Accutase passaging is that hPSC cultures are dissociated into single cell suspensions; this allows scale-up and applications such as transfection, FACS sorting, and cloning. Here we explain the appropriate hPSC feeder-free culture technique, recommend cell seeding densities, and show examples of colonies with good and bad morphology. Alternative feeder-free culture methods are discussed, such as alternative media, matrices, and passaging methods. We also provide a protocol for adapting hPSC cultures from feeder layers to feeder-free conditions, which can sometimes be a roadblock for many laboratories. Finally, we describe a method for the successful cryo-preservation of hPSCs cultured under feeder-free conditions.