Current Molecular Medicine (v.13, #5)

The reprogramming of somatic cells into induced pluripotent stem cells or iPS cells can be achievedby the ectopic expression of defined factors. Patient-specific iPS cell lines can be derived and used for diseasemodeling, 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 thisprocess. These methods include screening for new genetic factors and chemical compounds, and theengineering of new synthetic factors. Here, we review recent progress made in this field and show how a betterunderstanding of the ES (embryonic stem) cell transcriptional network is important for efficient reprogramming.

Pluripotent Stem Cell-Derived Somatic Stem Cells as Tool to Study the Role of MicroRNAs in Early Human Neural Development by B. Roese- Koerner, L. Stappert, P. Koch, O. Brustle, L. Borghese (707-722).
The in vitro differentiation of human pluripotent stem cells represents a convenient approach togenerate large numbers of neural cells for basic and translational research. We recently described thederivation of homogeneous populations of long-term self-renewing neuroepithelial-like stem cells from humanpluripotent stem cells (lt-NES® cells). These cells constitute a suitable source of neural stem cells for in vitromodelling of early human neural development. Recent evidence demonstrates that microRNAs are importantregulators of stem cells and nervous system development. Studies in several model organisms suggest thatmicroRNAs contribute to different stages of neurogenesis - from progenitor self-renewal to survival andfunction of differentiated neurons. However, the understanding of the impact of microRNA-based regulation inhuman neural development is still at its dawn. Here, we give an overview on the current state of microRNAbiology 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 asoverexpression 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 commitmentto neuronal differentiation. A detailed examination revealed that overexpression of miR-9 promotesdifferentiation, while overexpression of miR-9* affects both proliferation and differentiation of lt-NES® cells. Thiswork provides insights into the regulation of early human neuroepithelial cells by microRNAs and highlights thepotential of controlling differentiation of human stem cells by modulating the expression of selectedmicroRNAs.

Human Progenitor Cells for Bone Engineering Applications by G.M. de Peppo, P. Thomsen, C. Karlsson, R. Strehl, A. Lindahl, J. Hyllner (723-734).
In this report, the authors review the human skeleton and the increasing burden of bonedeficiencies, the limitations encountered with the current treatments and the opportunities provided by theemerging field of cell-based bone engineering. Special emphasis is placed on different sources of humanprogenitor cells, as well as their pros and cons in relation to their utilization for the large-scale construction offunctional bone-engineered substitutes for clinical applications. It is concluded that, human pluripotent stemcells represent a valuable source for the derivation of progenitor cells, which combine the advantages of bothembryonic and adult stem cells, and indeed display high potential for the construction of functional substitutesfor bone replacement therapies.

The Dark Side of Stem Cells: Triggering Cancer Progression by Cell Fusion by T. Dittmar, C. Nagler, B. Niggemann, K.S. Zanker (735-750).
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 placeduring pathophysiological processes such as virus entry into host cells and cancer. Particularly in cancer, cellfusion has been linked to a number of properties being associated with the progression of the diseaseincluding an increased proliferation rate, an enhanced metastatogenic behavior, an increased drug resistanceand an increased resistance towards apoptosis. Although the process of cell fusion including the molecules tobe involved-in is not completely understood in higher organisms, recent data revealed that chronicinflammation seems to be strong mediator. Since tumor tissue resembles chronically inflamed tissue, it can beconcluded 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 achronic inflamed microenvironment could originate in cancerous tissues, the role of M2-polarized tumorassociatedmacrophages (M2-TAMs) within this process and how fusion between macrophages and BMDCswill 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 betweentumor (stem) cells and BMDCs.

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

There are currently 1527 known microRNAs (miRNAs) in human, each of which may regulatehundreds 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 specificmiRNA. 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 themiRNA play key roles in regulating the production of mature miRNA. Mature miRNA enriched in stem cellscontrol stem cell self-renewal as well as their differentiation. Though specific miRNAs have been shown tocontrol differentiation towards various lineages such as neural or skin cells, some of the most wellcharacterized miRNAs have been found in promoting the formation of cardiac cells. In addition, miRNAs alsoplay a critical role in cardiomyocyte hypertrophy, especially in a pathological context. Such miRNAs arepredicted to be therapeutic targets for treating cardiovascular diseases. In this review we will discuss howmiRNAs act to maintain the stem cell state and also explore the current knowledge of the mechanisms thatregulate miRNAs. Furthermore, we will discuss the emerging roles of miRNAs using cardiomyocytedifferentiation and maturation as a paradigm. Emphasis will also be given on some of the less ventured areassuch as the role of miRNAs in the physiological maturation of cardiomyocytes. These potentially beneficialmiRNAs are likely to improve cardiac function in both in vivo and in vitro settings and thus provide additionalstrategy to treat heart diseases and more importantly serve as a good model for understanding cardiomyocytematuration in vitro.

Improved Generation of Patient-Specific Induced Pluripotent Stem Cells Using a Chemically-Defined and Matrigel-Based Approach by B. GroB, M. Sgodda, M. Rasche, A. Schambach, G. Gohring, B. Schlegelberger, B. Greber, T. Linden, D. Reinhardt, T. Cantz, J.-H. Klusmann (765-776).
Reprogramming of somatic cells into patient-specific pluripotent analogues of human embryonicstem cells (ESCs) emerges as a prospective therapeutic angle in molecular medicine and a tool for basic stemcell biology. However, the combination of relative inefficiency and high variability of non-defined cultureconditions precluded the use of this technique in a clinical setting and impeded comparability betweenlaboratories. To overcome these obstacles, we sequentially devised a reprogramming protocol using onelentiviral-based polycistronic reprogramming construct, optimized for high co-expression of OCT4, SOX2, KLF4and MYC in conjunction with small molecule inhibitors of non-permissive signaling cascades, such astransforming growth factor β (SB431542), MEK/ERK (PD0325901) and Rho-kinase signaling (Thiazovivin), in adefined extracellular environment. Based on human fetal liver fibroblasts we could efficiently derive inducedpluripotent 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 overallincrease 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 thisprotocol, we could derive iPSCs from patient fibroblasts, which were impermissive to classical reprogrammingefforts, and from a patient suffering from familial platelet disorder. Thus, our defined protocol for highly efficientreprogramming to generate patient-specific iPSCs, reflects a big step towards therapeutic and broad scientificapplication of iPSCs, even in previously unfeasible settings.

The problems of allocation of scarce resources and priority setting in health care have so far notbeen much studied in the context of stem cell-based therapeutic applications. If and when competitive costeffectivestem cell-based therapies are available, the problem of priority setting - to whom should stem cellbasedtherapies be offered and on what grounds - is discussed in this article using the examples ofParkinson's Disease (PD) and Huntington's Disease (HD). The aim of this paper is to examine the presentlyknown differences between PD and HD and analyze the role of these differences for setting priorities of stemcell-based therapeutic applications to treat these diseases. To achieve this aim, we (1) present the theoreticalframework used in the analysis; (2) compare PD and HD in terms of health related and non-health relatedconsequences of these diseases for patients, their relatives and third parties; (3) analyze the ethical relevanceof observed differences for priority setting given different values and variables; (4) compare PD and HD interms of social justice related consequences of stem cell-based therapies; and (5) analyze the ethicalrelevance of these differences for priority setting given different values and variables. We argue that the stepsof analysis applied in this paper could be helpful when setting priorities among treatments of other diseaseswith similar differences as those between PD and HD.

Human induced pluripotent stem cells (hiPSCs) have great potential as a robust source ofprogenitors for regenerative medicine. The novel technology also enables the derivation of patient-specificcells 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 embryonicstem cells (hESCs) is still unresolved. Variations among iPSCs, resulting from their original tissue or cellsource, and from the experimental protocols used for their derivation, significantly affect epigenetic propertiesand 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 theirparental tissue source. We also summarize the patient-specific iPSCs that have been derived for applicationsin biological research and drug discovery; and review risks that must be overcome in order to use iPSCtechnology for clinical applications.

What Makes a Pluripotency Reprogramming Factor? by R. Jauch, P.R. Kolatkar (806-814).
Resetting differentiated cells to a pluripotent state is now a widely applied technology and a key steptowards personalized cell replacement therapies. Conventionally, combinations of transcription factor proteinsare introduced into a differentiated cell to convert gene expression programs and to change cell fates. Yet, themolecular mechanism of nuclear reprogramming is only superficially understood. Specifically, it is unclear whatsets pluripotency reprogramming factors (PRFs) molecularly apart from other transcription factor moleculesthat induce, for example, lineage commitment in embryonic development. Ultimately, PRFs must scan thegenome of a differentiated cell, target enhancers of pluripotency factors and initiate gene expression. Thisrequires biochemical properties to selectively recognize DNA sequences, either alone or by cooperating withother 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 homologousyet functionally contrasting family members. Except for Klf4, the consensus DNA binding motifs are highlyconserved for PRFs when compared to non-pluripotency inducing family members, suggesting that theindividual DNA sequence preference may not be the distinguishing factor. By contrast, variant composite DNAmotifs were found in pluripotency enhancers that lead to a differential assembly of various Sox and Oct familymembers due selective protein-protein interaction platform. As a consequence, the cooperation of PRFs ondistinctly configured DNA motifs may underlie the reprogramming process. Indeed, it has been demonstratedthat Sox17 can be rationally engineered into a PRF by modulating its cooperation with Oct4. An in deepunderstanding of this phenomenon would allow rational engineering and optimization of PRFs. This way, thereprogramming efficiency can be enhanced and fine-tuned to generate optimal synthetic reagents forregenerative medicine.

Pluripotent stem cells hold great promise for future applications in many areas of regenerativemedicine. Their defining property of differentiation towards any of the three germ layers and all derivativesthereof, 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 towardshematopoietic 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 forregenerative purposes in clinical human medicine, namely in bone marrow transplantation, and also the firststem cells to be genetically altered for the first successful gene therapy trial in humans. However, because ofthe technical difficulties associated with this rare type of cell, such as the current incapability of prospectiveisolation, in vitro expansion and gene repair by homologous recombination, there is great interest in usingpluripotent stem cells, such as Embryonic Stem (ES-) cells, as a source for generating and genetically alteringHSCs, 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 anES-cell like state, so-called induced pluripotent stem (iPS) cells, this field of research has experienced a hugeboost. In this brief review, we will reflect on the status quo of directed hematopoietic differentiation of humanand mouse pluripotent stem cells.

Cumulative evidence shows that transplantation of stem cells (SC) derivatives can reduce thefunctional deficits induced by cerebral ischemia or hemorrhage in animals. Most SC sources have beenapplied 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 trialsdeveloped in parallel, and currently outnumber other applications of SC in neurological disorders. These trialsreflect a paradigm shift from cell replacement therapy to disease-modeling effects, with increased used of nonneuralSC. This shift stems in experimental demonstration of paracrine effects of SC that attenuateinflammation, limit cell death through neurotrophic effects, and enhance endogenous recovery processes. Dueto its pathogenic characteristics, stroke can uniquely benefit from this variety of actions.

Disease-Specific iPS Cell Models in Neuroscience by M. Peitz, J. Jungverdorben, O. Brustle (832-841).
Neurodegenerative diseases are a heterogeneous group of sporadic or familial disorders of thenervous system that mostly lead to a progressive loss of neural cells. A major challenge in studying themolecular pathomechanisms underlying these disorders is the limited experimental access to disease-affectedhuman nervous system tissue. In addition, considering that the molecular disease initiation occurs years ordecades before the symptomatic onset of a medical condition, these tissues mostly reflect only the final phaseof the disease. To overcome these limitations, various model systems have been established based on gainandloss-of-function studies in transformed cell lines or transgenic animal models. Although these approachesprovide valuable insights into disease mechanisms and development they often lack physiological proteinexpression levels and a humanized context of molecular interaction partners. The generation of humaninduced pluripotent stem (hiPS) cells from somatic cells provides access to virtually unlimited numbers ofpatient-specific cells for modeling neurological disorders in vitro. In this review, we focus on the currentprogress made in hiPS cell-based modeling of neurodegenerative diseases and discuss recent advances inthe quality assessment of hiPS cell lines.

Improved Hepatic Differentiation Strategies for Human Induced Pluripotent Stem Cells by M. Sgodda, S. Mobus, J. Hoepfner, A.D. Sharma, A. Schambach, B. Greber, M. Ott, T. Cantz (842-855).
Based on their almost unlimited self-renewal capacity and their ability to differentiate into derivativesof all three germ layers, human induced pluripotent stem cells (hiPSCs) might serve as a preferable source forhepatic transplants in metabolic liver disorders or acute liver failure. Furthermore, the generation of patientspecifichiPSCs might facilitate the development of innovative therapeutic strategies by accurately modellingdisease in vitro. In our study, we aimed for an efficient hepatic differentiation protocol that is applicable for bothhuman embryonic stem cells (hESCs) and hiPSCs. We attempted to accomplish this goal by using a cytokineand small molecule-based protocol for direct differentiation of hESCs and hiPSCs into hepatic cells. Selectingdifferentiated hepatic cells was possible using an albumin promoter-driven G418 resistance system. Due toIRES-dependent dTomato reporter expression, we were able to track hepatic differentiated cells and weevaluated the most efficient time frame for G418 selection. The status of hepatic differentiation was determinedby qRT-PCR comparing the expression of hepatic markers such as AFP, ALB, SOX17, and HNF4 to standardhepatic cells. Functional analysis of the hepatic phenotype was obtained by measuring secreted albumin levelsand by analysis of cytochrome P450 type 1A1 activity (EROD). The percentage of differentiated cells wasquantified by FACS analysis. In conclusion, our improved protocol demonstrates that both pluripotent cellsources (hESC and hiPSC) can efficiently be differentiated into mature hepatic cells with functionalcharacteristics similar to those of standard hepatic cell lines such as HepG2.

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

Recent reports demonstrate that the plasticity of mammalian somatic cells is much higher thanpreviously assumed and that ectopic expression of transcription factors may have the potential to induce theconversion 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 conversionof astrocytes into neurons or neural stem cells into monocytes has been demonstrated. Nowadays, in the eraof systems biology, continuously growing holistic data sets are providing increasing insights into coretranscriptional networks and cellular signaling pathways. This knowledge enables cell biologists to understandhow cellular fate is determined and how it could be manipulated. As a consequence for biomedicalapplications, it might be soon possible to convert patient specific somatic cells directly into desiredtransplantable other cell types. The clinical value, however, of such reprogrammed cells is currently limited dueto the invasiveness of methods applied to induce reprogramming factor activity. This review will focus onexperimental strategies to ectopically induce cell fate modulators. We will emphasize those strategies thatenable efficient and robust overexpression of transcription factors by minimal genetic alterations of the hostgenome. Furthermore, we will discuss procedures devoid of any genomic manipulation, such as the directdelivery of mRNA, proteins, or the use of small molecules. By this, we aim to give a comprehensive overviewon state of the art techniques that harbor the potential to generate safe reprogrammed cells for clinicalapplications.

New developments in DNA sequencing platforms and the advancements in GWAS studies(genome-wide association studies) are changing the understanding of human pathologies. Such developmentswill ultimately result in a deeper understanding of how genomic variations contribute to diseases.</p><p>Induced pluripotent stem cells (iPSCs) are currently entering clinical research phases, allowing theinvestigation 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 apatient; iPSC-derived cells can be used as a tool for drug screening or discovery. In combination with nextgeneration sequencing (NGS)-based and GWAS technologies, iPSCs have the potential to become a novelplatform technology to predict adverse drug and off-target effects, and using such cell models to predicttoxicity.</p><p>In view of the arising concepts of regenerative theranostics, iPSCs and NGS technologies provide a powerfulmeans to analyze the complexity of diseases on the molecular level and to better understand the processesthat lead to pathobiology.</p><p>To promote the widespread use of iPSC-based approaches in drug development it has to be shown that thecells can be reliably produced in the quantity, consistency and purity needed to meet pharmaceuticalstandards. Integrative genomics and genetic approaches have shown to be a useful tool in elucidating thecomplexity found in gene regulatory pathways. In this review, the application of pluripotent stem cells for thegeneration of next-generation theranostics and newer perspectives on iPSCs in modeling clinical diseases, arediscussed.