Current Molecular Medicine - Volume 13, Issue 5, 2013

The reprogramming of somatic cells into induced pluripotent stem cells or iPS cells can be achieved by the ectopic expression of defined factors. Patient-specific iPS cell lines can be derived and used for disease modeling, drug and toxicology screening, cellular replacement therapies and basic research. However, reprogramming is slow and inefficient and numerous methods have been described aiming to improve this process. These methods include screening for new genetic factors and chemical compounds, and the engineering of new synthetic factors. Here, we review recent progress made in this field and show how a better understanding of the ES (embryonic stem) cell transcriptional network is important for efficient reprogramming.

The in vitro differentiation of human pluripotent stem cells represents a convenient approach to generate large numbers of neural cells for basic and translational research. We recently described the derivation of homogeneous populations of long-term self-renewing neuroepithelial-like stem cells from human pluripotent stem cells (lt-NES® cells). These cells constitute a suitable source of neural stem cells for in vitro modelling of early human neural development. Recent evidence demonstrates that microRNAs are important regulators of stem cells and nervous system development. Studies in several model organisms suggest that microRNAs contribute to different stages of neurogenesis – from progenitor self-renewal to survival and function of differentiated neurons. However, the understanding of the impact of microRNA-based regulation in human neural development is still at its dawn. Here, we give an overview on the current state of microRNA biology in stem cells and neural development and examine the role of the neural-associated miR-124, miR- 125b and miR-9/9* in human lt-NES® cells. We show that overexpression of miR-124, as well as overexpression of miR-125b, impair lt-NES® cell self-renewal and induce differentiation into neurons. Overexpression of the miR-9/9* locus also impairs self-renewal of lt-NES® cells and supports their commitment to neuronal differentiation. A detailed examination revealed that overexpression of miR-9 promotes differentiation, while overexpression of miR-9* affects both proliferation and differentiation of lt-NES® cells. This work provides insights into the regulation of early human neuroepithelial cells by microRNAs and highlights the potential of controlling differentiation of human stem cells by modulating the expression of selected microRNAs.

In this report, the authors review the human skeleton and the increasing burden of bone deficiencies, the limitations encountered with the current treatments and the opportunities provided by the emerging field of cell-based bone engineering. Special emphasis is placed on different sources of human progenitor cells, as well as their pros and cons in relation to their utilization for the large-scale construction of functional bone-engineered substitutes for clinical applications. It is concluded that, human pluripotent stem cells represent a valuable source for the derivation of progenitor cells, which combine the advantages of both embryonic and adult stem cells, and indeed display high potential for the construction of functional substitutes for bone replacement therapies.

The phenomenon of cell fusion plays a crucial role in a plethora of physiological processes, including fertilization, wound healing, and tissue regeneration. In addition to this, cell fusion also takes place during pathophysiological processes such as virus entry into host cells and cancer. Particularly in cancer, cell fusion has been linked to a number of properties being associated with the progression of the disease including an increased proliferation rate, an enhanced metastatogenic behavior, an increased drug resistance and an increased resistance towards apoptosis. Although the process of cell fusion including the molecules to be involved-in is not completely understood in higher organisms, recent data revealed that chronic inflammation seems to be strong mediator. Since tumor tissue resembles chronically inflamed tissue, it can be concluded that cell fusion between recruited macrophages, bone marrow-derived cells (BMDCs), and tumor (stem) cells should be a common phenomenon in cancer. In the present review, we will summarize how a chronic inflamed microenvironment could originate in cancerous tissues, the role of M2-polarized tumorassociated macrophages (M2-TAMs) within this process and how fusion between macrophages and BMDCs will trigger cancer progression. A particular emphasis will be drawn on recurrence cancer stem cells (rCSCs), which will play a pivotal role in “oncogenic resistance” and which might originate from fusion events between tumor (stem) cells and BMDCs.

MicroRNAs (miRNAs) are de-regulated in cancer versus the normal tissue counterpart and actively participate in human carcinogenesis. Among the genes whose expression is under their control there are both oncogenes and tumor suppressor genes, revealing that it is not only limiting but simply wrong to assign them a function just as oncogenes or as tumor suppressor genes. In addition to primary tumors, miRNAs can be detected in almost all human body fluids and effectively help to diagnose cancer and to prognosticate clinical outcome and response to treatment of tumors. The advent of miRNA mimic and miRNA silencing molecules has allowed to modulate miRNA expression in tumors, showing that miRNAs can be effectively used as therapeutic agents. This review will focus on those findings that have provided the rationale for the use of miRNAs as patient “tailored” anti-cancer agents.

There are currently 1527 known microRNAs (miRNAs) in human, each of which may regulate hundreds or thousands of target genes. miRNA expression levels vary between cell types; for example, miR- 302 and miR-290 families are highly enriched in embryonic stem cells, while miR-1 is a muscle specific miRNA. miRNA biosynthesis and function are highly regulated and this regulation may be cell type specific. The processing enzymes and factors that recognize features in sequence and secondary structure of the miRNA play key roles in regulating the production of mature miRNA. Mature miRNA enriched in stem cells control stem cell self-renewal as well as their differentiation. Though specific miRNAs have been shown to control differentiation towards various lineages such as neural or skin cells, some of the most well characterized miRNAs have been found in promoting the formation of cardiac cells. In addition, miRNAs also play a critical role in cardiomyocyte hypertrophy, especially in a pathological context. Such miRNAs are predicted to be therapeutic targets for treating cardiovascular diseases. In this review we will discuss how miRNAs act to maintain the stem cell state and also explore the current knowledge of the mechanisms that regulate miRNAs. Furthermore, we will discuss the emerging roles of miRNAs using cardiomyocyte differentiation and maturation as a paradigm. Emphasis will also be given on some of the less ventured areas such as the role of miRNAs in the physiological maturation of cardiomyocytes. These potentially beneficial miRNAs are likely to improve cardiac function in both in vivo and in vitro settings and thus provide additional strategy to treat heart diseases and more importantly serve as a good model for understanding cardiomyocyte maturation in vitro.

Reprogramming of somatic cells into patient-specific pluripotent analogues of human embryonic stem cells (ESCs) emerges as a prospective therapeutic angle in molecular medicine and a tool for basic stem cell biology. However, the combination of relative inefficiency and high variability of non-defined culture conditions precluded the use of this technique in a clinical setting and impeded comparability between laboratories. To overcome these obstacles, we sequentially devised a reprogramming protocol using one lentiviral-based polycistronic reprogramming construct, optimized for high co-expression of OCT4, SOX2, KLF4 and MYC in conjunction with small molecule inhibitors of non-permissive signaling cascades, such as transforming growth factor β (SB431542), MEK/ERK (PD0325901) and Rho-kinase signaling (Thiazovivin), in a defined extracellular environment. Based on human fetal liver fibroblasts we could efficiently derive induced pluripotent stem cells (iPSCs) within 14 days. We attained efficiencies of up to 10.97±1.71% resulting in 79.5- fold increase compared to non-defined reprogramming using four singular vectors. We show that the overall increase of efficiency and temporal kinetics is a combinatorial effect of improved lentiviral vector design, signaling inhibition and definition of extracellular matrix (Matrigel®) and culture medium (mTESR®1). Using this protocol, we could derive iPSCs from patient fibroblasts, which were impermissive to classical reprogramming efforts, and from a patient suffering from familial platelet disorder. Thus, our defined protocol for highly efficient reprogramming to generate patient-specific iPSCs, reflects a big step towards therapeutic and broad scientific application of iPSCs, even in previously unfeasible settings.

The problems of allocation of scarce resources and priority setting in health care have so far not been much studied in the context of stem cell-based therapeutic applications. If and when competitive costeffective stem cell-based therapies are available, the problem of priority setting - to whom should stem cellbased therapies be offered and on what grounds - is discussed in this article using the examples of Parkinson's Disease (PD) and Huntington's Disease (HD). The aim of this paper is to examine the presently known differences between PD and HD and analyze the role of these differences for setting priorities of stem cell-based therapeutic applications to treat these diseases. To achieve this aim, we (1) present the theoretical framework used in the analysis; (2) compare PD and HD in terms of health related and non-health related consequences of these diseases for patients, their relatives and third parties; (3) analyze the ethical relevance of observed differences for priority setting given different values and variables; (4) compare PD and HD in terms of social justice related consequences of stem cell-based therapies; and (5) analyze the ethical relevance of these differences for priority setting given different values and variables. We argue that the steps of analysis applied in this paper could be helpful when setting priorities among treatments of other diseases with similar differences as those between PD and HD.

Human induced pluripotent stem cells (hiPSCs) have great potential as a robust source of progenitors for regenerative medicine. The novel technology also enables the derivation of patient-specific cells for applications to personalized medicine, such as for personal drug screening and toxicology. However, the biological characteristics of iPSCs are not yet fully understood and their similarity to human embryonic stem cells (hESCs) is still unresolved. Variations among iPSCs, resulting from their original tissue or cell source, and from the experimental protocols used for their derivation, significantly affect epigenetic properties and differentiation potential. Here we review the potential of iPSCs for regenerative and personalized medicine, and assess their expression pattern, epigenetic memory and differentiation capabilities in relation to their parental tissue source. We also summarize the patient-specific iPSCs that have been derived for applications in biological research and drug discovery; and review risks that must be overcome in order to use iPSC technology for clinical applications.

Resetting differentiated cells to a pluripotent state is now a widely applied technology and a key step towards personalized cell replacement therapies. Conventionally, combinations of transcription factor proteins are introduced into a differentiated cell to convert gene expression programs and to change cell fates. Yet, the molecular mechanism of nuclear reprogramming is only superficially understood. Specifically, it is unclear what sets pluripotency reprogramming factors (PRFs) molecularly apart from other transcription factor molecules that induce, for example, lineage commitment in embryonic development. Ultimately, PRFs must scan the genome of a differentiated cell, target enhancers of pluripotency factors and initiate gene expression. This requires biochemical properties to selectively recognize DNA sequences, either alone or by cooperating with other PRFs. In this review, we will discuss the molecular make-up of the prominent PRFs Sox2, Oct4, Klf4, Esrrb, Nr5a2 and Nanog and attempt to identify unique features distinguishing them from highly homologous yet functionally contrasting family members. Except for Klf4, the consensus DNA binding motifs are highly conserved for PRFs when compared to non-pluripotency inducing family members, suggesting that the individual DNA sequence preference may not be the distinguishing factor. By contrast, variant composite DNA motifs were found in pluripotency enhancers that lead to a differential assembly of various Sox and Oct family members due selective protein-protein interaction platform. As a consequence, the cooperation of PRFs on distinctly configured DNA motifs may underlie the reprogramming process. Indeed, it has been demonstrated that Sox17 can be rationally engineered into a PRF by modulating its cooperation with Oct4. An in deep understanding of this phenomenon would allow rational engineering and optimization of PRFs. This way, the reprogramming efficiency can be enhanced and fine-tuned to generate optimal synthetic reagents for regenerative medicine.

Pluripotent stem cells hold great promise for future applications in many areas of regenerative medicine. Their defining property of differentiation towards any of the three germ layers and all derivatives thereof, including somatic stem cells, explains the special interest of the biomedical community in this cell type. In this review, we focus on the current state of directed differentiation of pluripotent stem cells towards hematopoietic stem cells (HSCs). HSCs are especially interesting because they are the longest known and, thus, most intensively investigated somatic stem cells. They were the first stem cells successfully used for regenerative purposes in clinical human medicine, namely in bone marrow transplantation, and also the first stem cells to be genetically altered for the first successful gene therapy trial in humans. However, because of the technical difficulties associated with this rare type of cell, such as the current incapability of prospective isolation, in vitro expansion and gene repair by homologous recombination, there is great interest in using pluripotent stem cells, such as Embryonic Stem (ES-) cells, as a source for generating and genetically altering HSCs, ex vivo. This has been hampered by ethical concerns associated with the use of human ES-cells. However, since Shinya Yamanaka´s successful attempts to reprogram somatic cells of mice and men to an ES-cell like state, so-called induced pluripotent stem (iPS) cells, this field of research has experienced a huge boost. In this brief review, we will reflect on the status quo of directed hematopoietic differentiation of human and mouse pluripotent stem cells.

Cumulative evidence shows that transplantation of stem cells (SC) derivatives can reduce the functional deficits induced by cerebral ischemia or hemorrhage in animals. Most SC sources have been applied to stroke models, with varying degrees of differentiation into neural derivatives and in varying number, timing and route of administration, with similar benefits on functional outcome. Pioneering clinical trials developed in parallel, and currently outnumber other applications of SC in neurological disorders. These trials reflect a paradigm shift from cell replacement therapy to disease-modeling effects, with increased used of nonneural SC. This shift stems in experimental demonstration of paracrine effects of SC that attenuate inflammation, limit cell death through neurotrophic effects, and enhance endogenous recovery processes. Due to its pathogenic characteristics, stroke can uniquely benefit from this variety of actions.

Neurodegenerative diseases are a heterogeneous group of sporadic or familial disorders of the nervous system that mostly lead to a progressive loss of neural cells. A major challenge in studying the molecular pathomechanisms underlying these disorders is the limited experimental access to disease-affected human nervous system tissue. In addition, considering that the molecular disease initiation occurs years or decades before the symptomatic onset of a medical condition, these tissues mostly reflect only the final phase of the disease. To overcome these limitations, various model systems have been established based on gainand loss-of-function studies in transformed cell lines or transgenic animal models. Although these approaches provide valuable insights into disease mechanisms and development they often lack physiological protein expression levels and a humanized context of molecular interaction partners. The generation of human induced pluripotent stem (hiPS) cells from somatic cells provides access to virtually unlimited numbers of patient-specific cells for modeling neurological disorders in vitro. In this review, we focus on the current progress made in hiPS cell-based modeling of neurodegenerative diseases and discuss recent advances in the quality assessment of hiPS cell lines.

Based on their almost unlimited self-renewal capacity and their ability to differentiate into derivatives of all three germ layers, human induced pluripotent stem cells (hiPSCs) might serve as a preferable source for hepatic transplants in metabolic liver disorders or acute liver failure. Furthermore, the generation of patientspecific hiPSCs might facilitate the development of innovative therapeutic strategies by accurately modelling disease in vitro. In our study, we aimed for an efficient hepatic differentiation protocol that is applicable for both human embryonic stem cells (hESCs) and hiPSCs. We attempted to accomplish this goal by using a cytokine and small molecule-based protocol for direct differentiation of hESCs and hiPSCs into hepatic cells. Selecting differentiated hepatic cells was possible using an albumin promoter-driven G418 resistance system. Due to IRES-dependent dTomato reporter expression, we were able to track hepatic differentiated cells and we evaluated the most efficient time frame for G418 selection. The status of hepatic differentiation was determined by qRT-PCR comparing the expression of hepatic markers such as AFP, ALB, SOX17, and HNF4 to standard hepatic cells. Functional analysis of the hepatic phenotype was obtained by measuring secreted albumin levels and by analysis of cytochrome P450 type 1A1 activity (EROD). The percentage of differentiated cells was quantified by FACS analysis. In conclusion, our improved protocol demonstrates that both pluripotent cell sources (hESC and hiPSC) can efficiently be differentiated into mature hepatic cells with functional characteristics similar to those of standard hepatic cell lines such as HepG2.

Cell therapy with mesenchymal stromal cells (MSCs) is the focus of intensive investigation. Several clinical trials, including large-scale placebo-controlled phase III clinical trials, are currently underway evaluating the therapeutic potential of autologous and allogeneic MSCs for treatment of catastrophic inflammatory diseases, including steroid-refractory graft-versus-host disease (GvHD), multiple sclerosis (MS) and Crohn’s disease. MSCs are also being investigated as carriers of anti-cancer biotherapeutics. We here review recent developments in our understanding of the immunosuppressive properties of MSCs. We firstly discuss the effects of ex vivo culture conditions on the phenotype and functions of MSCs. Secondly, we summarize the immune functions suppressed by MSCs with a focus on T cell, B cell, natural killer cell and dendritic cell functions. Thirdly, we discuss newly identified pathways responsible for the immunosuppressive activity of MSCs, including the expression of heme-oxygenase (HO)-1, the secretion of galectins, CCL2 antagonism, T regulatory cell (Treg) cross-talk and production of TNF-α stimulated gene/protein-6 (TSG-6). Finally, we review the literature on the molecular pathways governing MSC homing and discuss recent clinical data on the use of MSCs for treatment of GvHD, MS and Crohn’s disease.

Recent reports demonstrate that the plasticity of mammalian somatic cells is much higher than previously assumed and that ectopic expression of transcription factors may have the potential to induce the conversion of any cell type into another. Fibroblast cells can be converted into embryonic stem cell-like cells, neural cells, cardiomyocytes, macrophage-like cells as well as blood progenitors. Additionally, the conversion of astrocytes into neurons or neural stem cells into monocytes has been demonstrated. Nowadays, in the era of systems biology, continuously growing holistic data sets are providing increasing insights into core transcriptional networks and cellular signaling pathways. This knowledge enables cell biologists to understand how cellular fate is determined and how it could be manipulated. As a consequence for biomedical applications, it might be soon possible to convert patient specific somatic cells directly into desired transplantable other cell types. The clinical value, however, of such reprogrammed cells is currently limited due to the invasiveness of methods applied to induce reprogramming factor activity. This review will focus on experimental strategies to ectopically induce cell fate modulators. We will emphasize those strategies that enable efficient and robust overexpression of transcription factors by minimal genetic alterations of the host genome. Furthermore, we will discuss procedures devoid of any genomic manipulation, such as the direct delivery of mRNA, proteins, or the use of small molecules. By this, we aim to give a comprehensive overview on state of the art techniques that harbor the potential to generate safe reprogrammed cells for clinical applications.

New developments in DNA sequencing platforms and the advancements in GWAS studies (genome-wide association studies) are changing the understanding of human pathologies. Such developments will ultimately result in a deeper understanding of how genomic variations contribute to diseases. Induced pluripotent stem cells (iPSCs) are currently entering clinical research phases, allowing the investigation of disease pathways and the identification of new targets and potentially druggable biomarkers. IPSCs can serve as a model for studying human diseases as they retain all the genetic information from a patient; iPSC-derived cells can be used as a tool for drug screening or discovery. In combination with next generation sequencing (NGS)-based and GWAS technologies, iPSCs have the potential to become a novel platform technology to predict adverse drug and off-target effects, and using such cell models to predict toxicity. In view of the arising concepts of regenerative theranostics, iPSCs and NGS technologies provide a powerful means to analyze the complexity of diseases on the molecular level and to better understand the processes that lead to pathobiology. To promote the widespread use of iPSC-based approaches in drug development it has to be shown that the cells can be reliably produced in the quantity, consistency and purity needed to meet pharmaceutical standards. Integrative genomics and genetic approaches have shown to be a useful tool in elucidating the complexity found in gene regulatory pathways. In this review, the application of pluripotent stem cells for the generation of next-generation theranostics and newer perspectives on iPSCs in modeling clinical diseases, are discussed.