Expression profile of the stem cell markers in human Hertwig’s epithelial root sheath/Epithelial rests of Malassez cells

Hertwig’s epithelial root sheath/Epithelial rests of Malassez (HERS/ERM) cells are unique epithelial cells in the periodontal ligament. They remain in periodontal tissues through-out the adult life, and it is expected that their functional role is to maintain the homeostasis of the periodontium through reciprocal interactions with other periodontal cells. In this study, we investigated whether HERS/ERM cells have primitive stem cell characteristics: those of embryonic stem cells as well as of epithelial stem cells. Primary HERS/ERM cells had typical epithelial cell morphology and characteristics and they maintained for more than five passages. They expressed epithelial stem cell-related genes: ABCG2, ANp63, p75, EpCAM, and Bmi-1. Moreover, the expression of embryonic stem cell markers such as Oct-4, Nanog, and SSEA-4 were detected. Next, we investigated whether the expression of these stem cell markers was maintained during the sub-culture process. HERS/ERM cells showed different expression levels of these stemness genes at each passage, but their expression was maintained throughout the passages. Taken together, our data suggest that a primary culture of HERS/ERM cells contains a population of primitive stem cells that express epithelial stem cell markers and embryonic stem cell markers. Furthermore, these cell populations were maintained during the sub-culturing process in our culture conditions. Therefore, our findings suggest that there is a strong possibility of accomplishing cementum tissue engineering with HERS/ERM cells.     

NG2-glia as multipotent neural stem cells: fact or fantasy?

Cycling glial precursors-"NG2-glia"-are abundant in the developing and mature central nervous system (CNS). During development, they generate oligodendrocytes. In culture, they can revert to a multipotent state, suggesting that they might have latent stem cell potential that could be harnessed to treat neurodegenerative disease. This hope has been subdued recently by a series of fate-mapping studies that cast NG2-glia as dedicated oligodendrocyte precursors in the healthy adult CNS-though rare, neuron production in the piriform cortex remains a possibility. Following CNS damage, the repertoire of NG2-glia expands to include Schwann cells and possibly astrocytes-but so far not neurons. This reaffirms the central role of NG2-glia in myelin repair. The realization that oligodendrocyte generation continues throughout normal adulthood has seeded the idea that myelin genesis might also be involved in neural plasticity. We review these developments, highlighting areas of current interest, contention, and speculation.     

Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase–AIF pathway

PADs (peptidylarginine deiminases) are calcium-dependent enzymes that change protein-bound arginine to citrulline (citrullination/deimination) affecting protein conformation and function. PAD up-regulation following chick spinal cord injury has been linked to extensive tissue damage and loss of regenerative capability. Having found that human neural stem cells (hNSCs) expressed PAD2 and PAD3, we studied PAD function in these cells and investigated PAD3 as a potential target for neuroprotection by mimicking calcium-induced secondary injury responses. We show that PAD3, rather than PAD2 is a modulator of cell growth/death and that PAD activity is not associated with caspase-3-dependent cell death, but is required for AIF (apoptosis inducing factor)-mediated apoptosis. PAD inhibition prevents association of PAD3 with AIF and AIF cleavage required for its translocation to the nucleus. Finally, PAD inhibition also hinders calcium-induced cytoskeleton disassembly and association of PAD3 with vimentin, that we show to be associated also with AIF; together this suggests that PAD-dependent cytoskeleton disassembly may play a role in AIF translocation to the nucleus. This is the first study highlighting a role of PAD activity in balancing hNSC survival/death, identifying PAD3 as an important upstream regulator of calcium-induced apoptosis, which could be targeted to reduce neural loss, and shedding light on the mechanisms involved.     

Nuclear receptors and microRNAs: Who regulates the regulators in neural stem cells?

In this mini-review, we focus on regulatory loops between nuclear receptors and microRNAs, an emerging class of small RNA regulators of gene expression. Evidence supporting interactions between microRNAs and nuclear receptors in the regulation of gene expression networks is discussed in relation to its possible role in neural stem cell self renewal and differentiation. Furthermore, we discuss possible disturbances of the regulatory loops between microRNAs and nuclear receptors in human neurodegenerative disease. Finally, we discuss the possible use of nuclear receptors as pharmacological entry points to regulate neural stem cell self-renewal and differentiation.     

New frontiers in human cell biology and medicine: Can pluripotent stem cells deliver?

Human pluripotent stem cells provide enormous opportunities to treat disease using cell therapy. But human stem cells can also drive biomedical and cell biological discoveries in a human model system, which can be directly linked to understanding disease or developing new therapies. Finally, rigorous scientific studies of these cells can and should inform the many science and medical policy issues that confront the translation of these technologies to medicine. In this paper, I discuss these issues using amyotrophic lateral sclerosis as an example.Much of modern cell biological discovery has been driven by the study of a diverse variety of primary and transformed cultured cells. However, the advent of human pluripotent cells is providing new avenues of discovery. These cells are genetically manipulable, euploid, expandable to large numbers, and can differentiate to most if not all human cell types. In addition, these cells can be used to analyze the large number of mutations and diverse genetic variation present in large human populations. In fact, not only are human pluripotent stem cells useful for typical cell culture experiments, but they are amenable to many of the types of genetic and molecular genetic approaches that historically have only been feasible in genetic and developmental systems, such as Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, or mouse.There are two types of human pluripotent stem cells in use. Human embryonic stem cells (hESC) are derived from human embryos that would otherwise be discarded and that are generally donated with substantial informed consent and ethical requirements (Shamblott et al., 1998; Thomson et al., 1998). Human induced pluripotent stem cells (hIPSC) are generated by several different reprogramming technologies, generally from fibroblasts obtained from small skin biopsies or other human somatic cell types, such as blood (Takahashi et al., 2007). Recent work suggests that hESC and hIPSC, although not identical in their properties, share very important features. First, hESC and hIPSC are both pluripotent so that any cell type of interest can in principle be generated. In fact, differentiation methods for many types of specialized human cells are being developed rapidly, fueled in large part by the need to generate stable differentiated derivatives for cell therapy. Second, hIPSC and hESC can be handled in the laboratory under conditions that are relatively straightforward for skilled cell culture scientists. Third, both hIPSC and hESC, when handled properly, are genetically relatively stable with a diploid karyotype so that gene dose and gene expression at all loci is effectively “normal” or at least representative of expression levels in cells in the intact human. These properties make these cells or their differentiated derivatives suitable for genetic screens using RNA interference, small molecules, insertional mutagenesis, or other analogous tools. Important differences between hESC and hIPSC include an apparent elevation in mutation load in hIPSC (Gore et al., 2011) and differences in epigenetic state. Either of these features may substantially influence the behavior of each cell type and its differentiated derivatives. Thus, hESC and hIPSC may play different roles in the discovery of new cellular and disease mechanisms and in the development of cellular therapies. Recent examples of novel discoveries made using hESC and hIPSC include substantial new insights into the control of the pluripotent state itself and identification of molecular pathways controlling cellular differentiation.

Disease in a dish approaches with hESC and hIPSC

Although hESC and hIPSC are just beginning to be used to probe and elucidate new cellular processes, there is already substantial progress using pluripotent stem cell approaches to unravel disease mechanisms using so-called disease in a dish paradigms (Unternaehrer and Daley, 2011). Disease in a dish methods use gene manipulation and/or reprogramming technologies to generate hESC or hIPSC lines with genomes carrying known mutations causing human disease, lesions such as shRNA expression mimicking human disease mutations (Marchetto et al., 2010; Ordonez et al., 2012), or genomes carrying combinations of known or unknown variants that contribute to disease (Fig. 1). With the advent of powerful molecular tools, such as Tal effector nucleases, individual genes can be manipulated by introducing point mutations with great precision (Hockemeyer et al., 2011). Thus, disease-causing mutations or other genetic lesions can be studied for their impacts on cellular processes under “normal” conditions of gene expression and in different genetic backgrounds. Similarly, suppressor and enhancer studies are feasible and will help unravel poorly understood cellular mechanisms. Finally, after differentiation to specialized cell types, cellular mechanisms and interactions as well as disease and potential therapies can be evaluated in bona fide human cells. These approaches are in their infancy but have substantial potential given the limitations of mouse models of disease to accurately recapitulate human disease and the many obvious differences between the details of mouse physiology and human physiology. They also bring unique advantages for diseases in which the key cell types, e.g., human central nervous system neurons, are difficult if not impossible to obtain in good condition or early in disease.Open in a separate windowFigure 1.Disease in a dish. Stem cells can be used to analyze how human genetic variation or mutation contributes to defined cellular phenotypes. Using neuronal phenotype as an example, Tal effector nucleases (TALENs) can be used to make defined changes in hIPSC of a common genetic background to analyze the contribution of a defined mutation or more complex variation to neuronal phenotypes.Examples of recent disease in dish studies include a variety of neurodegenerative diseases, heart diseases, and diseases of other organ systems (Unternaehrer and Daley, 2011). Although this work is in its early stages, there is a great deal of potential for meaningful mechanistic cell biological research in this collection of intriguing areas. In addition, these disease in dish models provide unique human materials for direct testing of drug safety and efficacy.

Amyotrophic lateral sclerosis (ALS): A disease in a dish paradigm

ALS, also known as Lou Gehrig’s disease, provides an intriguing example that illustrates the path from mechanism-based research to understanding and potential therapies using disease in a dish approaches. ALS is a disease in which death of motor neurons causes paralysis of voluntary muscles. As the disease progresses, paralysis ultimately extends to the muscles involved in breathing, swallowing, and all other voluntary movements. Once diagnosed, ALS generally causes death within 3–5 yr or less. There is only one approved drug for ALS, riluzole, but individual patients do not perceive much benefit because riluzole generates statistical prolongation of life for only a few months based on large-scale clinical trials. The key problem of course is to learn what causes death of motor neurons in ALS and whether this information might help develop a therapy that protects motor neurons from dysfunction and death.Although most ALS is “sporadic,” some forms are hereditary, including a form caused by mutations in the gene encoding dismutase SOD1 (superoxide 1). The generation of transgenic mouse and rat models that carry human mutant SOD1 genes has led to substantial progress in the understanding of cellular mechanisms that contribute to disease. In particular, a series of genetic studies in transgenic and chimeric mice led to the realization that the death of motor neurons in ALS might not be cell autonomous (Clement et al., 2003; Boillée et al., 2006; Yamanaka et al., 2008a,b). Disease in a dish studies using astrocytes carrying SOD1 mutant genes mixed with in vitro differentiated motor neurons made from pluripotent stem cells confirmed these findings and lead directly to searches for secreted toxic factors and drug testing (Di Giorgio et al., 2007, 2008; Marchetto et al., 2008). The general conclusion from these studies is that motor neuron death in ALS is strongly influenced by other cells in the spinal cord that make important contributions to, or protect from, motor neuron death. Whether astrocytes, microglia, or other cell types carry mutant SOD1 genes determines whether they exhibit stimulatory or protective effects on motor neuron death. Although such conclusions might be limited to SOD1-mediated ALS, there is also recent evidence that astrocytes might contribute to sporadic ALS as well (Haidet-Phillips et al., 2011).

Development of cell replacement or augmentation therapies

Successfully treating debilitating and currently incurable diseases with cell replacement or augmentation therapies requires basic cell biological research to fuel the generation and testing of new therapies (Fig. 2). For example, expansion of hematopoietic stem cell therapeutic approaches from leukemias to other diseases is based on a sound understanding of the basic cell and developmental biology of these cells. Several different stem cell therapies are in the midst of development and testing for several disorders, including spinal cord injury, graft versus host disease, skin diseases, blindness, diabetes, and AIDS (e.g., California Institute for Regenerative Medicine, 2009; Pollack, 2012; Schwartz et al., 2012). Using ALS as an example, some stem cell–based therapy efforts are aimed at trying to replace motor neurons that are damaged or die in ALS. But inducing motor neurons or their stem cell precursors to engraft into spinal cords or upper motor cortex and then appropriately extend axons and wire to peripheral muscles may be a challenge that will take many years to solve. Interestingly, the evidence that ALS is non–cell autonomous with major contributions from astrocytes and other glia has led to two different categories of cell therapy approach. One approach, which I regard as little more than guesswork, has tried to treat ALS by introducing poorly defined mesenchymal stem cells or cord blood stem cells directly into the spinal cord of ALS model animals. In fact, even in the absence of strong and reliable evidence, a clinical trial of cord blood stem cells transplanted into the spinal cord of human ALS patients was launched by a private company (http://www.tcacellulartherapy.com/fda_clinical_trials.html) and then halted by the FDA. A more rational approach given the state of scientific understanding, the state of experiments in animal models, and the in vitro data is to introduce progenitor cells that can differentiate to astrocytes or progenitors that secrete growth factors (Klein et al., 2005; Suzuki et al., 2007; Lepore et al., 2008; Suzuki and Svendsen, 2008; Hefferan et al., 2012). One of these approaches has recently reached clinical trials using fetal-derived spinal cord stem cells in which one hopes that enough will be learned to support more trials using different stem cell–generated preparations and perhaps different surgical methods or spinal cord sites.Open in a separate windowFigure 2.Cell replacement therapy using stem cells. A possible path from patients with hereditary ALS to cell biological research using transgenic mouse or pluripotent stem cell models to cell therapies. In this example, assessing the contribution of different cell types to ALS through rigorous research can lead to a rational approach to cell therapy.

Driving evidence-based scientific and medical policy with stem cell–driven discovery

The social and medical issues that arise in the development of cell therapies are and will be heavily influenced by the scientific discoveries about and using human stem cells. These social and medical challenges are well illustrated by a discussion of ALS owing to its rapidly progressive and devastating nature.First is whether one type of therapy can treat all types of ALS patients. Solving this issue will require a better understanding of what causes ALS, what cellular mechanisms contribute to motor neuron death, and which cells contribute in different forms of ALS. One key and possibly false assumption that drives current efforts is that all forms of ALS will exhibit the type of cellular nonautonomy found in animal models of SOD1-mediated ALS. Thus, models of sporadic ALS and hereditary forms of ALS such as those mediated by FUS/TLS or TDP-43 mutations must be tested. These experiments will also allow tests of the magnitude of the relative contributions of different cell types to motor neuron death or rescue in different forms of ALS. Additionally, if the actual cellular pathways that are defective in astrocytes and motor neurons can be better defined, cellular augmentation strategies and drug discovery could take advantage of that information.Second is the so-called snake oil problem (http://www.closerlookatstemcells.org; CBSNews, 2010). Numerous misleading and probably fraudulent advertisements can be found about clinics offering stem cell cures for ALS. These wild claims ignore large amounts of scientific data about the nature of ALS and rational approaches to therapy and prey upon those who don’t have ready access to or cannot evaluate legitimate scientific and medical information. Our legitimate scientific and medical community needs to stand against these frauds and provide accurate information derived from rigorous research to patients so that they are not taken advantage of by these clinics. In addition, we must work to ensure that legitimate efforts are not damaged by the blowback from those who effectively steal from desperately ill patients and their families or the likely harm to these patients that is coming from clinics that dispense untested and sometimes dangerous therapies.Third is the cell tracking problem. Currently, it is difficult to know how cells transplanted into the spinal cord of an ALS patient behave until after a patient has died. In addition, using antibodies to examine postmortem material from a transplant patient is problematic because the transplants are of human cells into a human patient. We desperately need to develop safe and sensitive methods for cell marking and imaging that will allow us to track cell behavior in patients in real time after transplant. Real-time measures would allow therapy to be modified or even repeated based on the analysis of cell behavior. These methods will rely heavily on cell biological research to identify cellular pathways and markers that could be visualized in real time by magnetic resonance imaging, positron emission tomography, or other imaging modalities.Fourth is how to manage individual patient response versus the average response of patients in a clinical trial. Although most often thought about with respect to drug therapies, different forms of ALS might vary in their response to cell therapies. An interesting possibility is that hIPSC lines from individual patients could be used not only for drug testing but also for evaluating the genetically driven contribution of different cell types to each patient’s version of ALS. A corollary is that for ALS patients included in a clinical trial, the notion that cells would be introduced only once and that the patient would then be “passively” followed with no change in treatment paradigm until death might be unacceptable. In conventional medicine, one might try treatment again or modify treatment course, depending on how an individual patient responds. On the other hand, prospective design of a statistically rigorous clinical trial requires development of a treatment plan and identification of rigorous outcome measures that should not be modified if the data are to be interpretable. Development of new statistical methods, outcome measures, hIPSC evaluation of phenotypes, and perhaps, cellular marking methods might allow trials to tolerate modification as part of an ALS patient’s clinical care. Perhaps rigorous data from hIPSC-based research could be used to make a case to the FDA that ALS clinical trials need to be more responsive to patient needs and variable outcomes with a disease that is as complicated and clinically inconsistent as ALS.Fifth, and finally, is the risk benefit analysis that can hinder or accelerate the development of therapies for rapidly fatal diseases such as ALS. Current paradigms of therapy development are risk averse and require enormous amounts of information on safety and possible efficacy before trials can be approved, financed, and launched. Yet, some patient populations, such as those with ALS, when facing a near certain death sentence, are very risk tolerant and might be willing to participate in trials with much lower certainty. Our community must work with the FDA to tackle this problem and to perhaps dramatically accelerate the introduction of good, but perhaps radical, ideas that might work but in which safety or efficacy testing in animals could take many years, or simply be unreliable, so that current patients would have no hope of benefiting. I often ask myself, as I work with my colleagues to develop a cell-based therapy for ALS that has been partially tested in animals but is not yet complete and therefore not ready for humans, what I would do if I, my wife, or one of my children developed ALS. Would I be willing to have appropriate types of stem cells or their derivatives transplanted into them even if I were not absolutely certain and had not yet proven absolute safety or efficacy? Interestingly, I find that in thinking about this issue, I fall back on my scientific understanding of ALS and the rigorous types of data on ALS mechanisms in the mainstream scientific literature. The result is that my own risk tolerance rises substantially when I have the ability to consider published and unpublished data and how it might be applied. I think that all ALS patients should have this information and that the FDA should be responsive to these patients when they want to take well-informed risks with experimental therapies that may not yet meet current FDA standards. Clearly, the devil will be in the details for implementing such an approach and ensuring patient protection as well as opportunity, but we owe this consideration to current ALS patients and those with comparably severe diseases. Again, however, this is a debate in which rigorous scientific research can drive the agenda and resulting policies.

Concluding remarks

Virchow developed the concept that disease arises in the individual cells of a tissue (Schultz, 2008). This important principle is the foundation for using human stem cells to understand basic cellular mechanisms and to extend that understanding to the development of therapies. Treating disease by targeting the misbehaving cells is clearly a wonderful opportunity for therapy development and research. Thus, probing the secrets of human cells by taking advantage of human pluripotent stem cells may signal the dawn of a new era in cell biology research.Finally, consider the many remarkable discoveries and novel mechanisms found when the basic tools of cell and molecular biology were applied to unusual members of the model organism toolbox, including snakes, ciliates, planaria, jerboa, and other organisms that have developed unusual biological strategies during evolution. Could humans be added to this list, and could the study of basic human cell biology yield comparable discoveries? Because humans are a large, long-lived organism with a complex brain, a rich evolutionary history, and substantial genetic variation across large and accessible populations, the answer is certain to be yes.     

Influence of interferon-α on the expression of the cancer stem cell markers in pancreatic carcinoma cells

The cytokine interferon-α (IFNα) belongs to the group of type I interferons already used in cancer therapy. This drug possesses radio- and chemo-sensitizing, and shows anti-angiogenic properties. Cancer stem cells (CSC) are a unique population of tumor cells that initiate secondary tumors, and are responsible for metastasis formation. Patients with pancreatic ductal adenocarcinoma (PDAC) have an especially poor prognosis, with 5-year survival rates of only ~1% and median survival of 4–6 months. PDAC is characterized by the presence of CSC. In this work we demonstrate for the first time that IFNα up-regulates the expression of the CSC markers CD24, CD44 and CD133 in in vitro and in vivo models of PDAC. We showed the IFNα effects on the migration and invasion of PDAC cells, which is associated with the level of the CSC marker expression. In vivo, this drug inhibits tumor growth but promotes metastasis formation in the early stage of tumor growth. We propose that IFNα may enhance the enrichment of CSC in PDAC tumors. Additionally we also suggest that in combination therapy of solid tumors with IFNα, this drug should be given to patients prior to chemotherapy to achieve the CSC activation.     

Role of miRNA-146?in proliferation and differentiation of mouse neural stem cells

Neural stem cells (NSCs) have been defined as neural cells with the potential to self-renew and eventually generate all cell types of the nervous system. NSCs serve as an ideal cell type for nervous system repair. In the present study, miR-146 overexpression and predicted target (notch 1) were used to study proliferation and differentiation of mouse NSCs. shRNA were used to demonstrate the function of Notch 1 in proliferation of mouse NSCs and luciferase reporter assay was used to assess and confirm the binding sequence of 3′-UTR between Notch 1 and miR-146. Results showed that miR-146 overexpression and knockdown of notch 1 inhibited proliferation of mouse NSCs under serum-free cultural conditions and promoted spontaneous differentiation of mouse NSCs under contained serum cultural conditions respectively. Mouse NSCs spontaneously underwent differentiation into neurogenic cells with contained serum medium. However, when miR-146 was overexpressed, differentiation efficiency of glial cells from NSCs was increased, suggesting that Notch1 promoted NSC proliferation and repressed spontaneous differentiation of NSC in serum-free medium. In conclusion, our results demonstrate that miR-146 promoted spontaneous differentiation of NSCs, and this mechanism was influenced by miR-146, as well as its target (notch 1) and downstream gene.     

Expression of TGFβ family factors and FGF2 in mouse and human embryonic stem cells maintained in different culture systems

Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases of pluripotency, we examined the expression of TGFβ family factors (ActivinA, Nodal, Lefty1, TGFβ1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFβ1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFβ1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3 and BMP/Smad1/5/8 endogenous branches of TGFβ signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFβ family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smad1/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primed states of pluripotency demonstrate diverse involvement of this factor in the regulation of the pluripotent cell self-renewal.     

Is hypothermia a stress condition in HepG2 cells? Expression and localization of Hsp70 in human hepatoma cell line

To understand hypothermia as a stress condition we determined the expression and localization of Hsp70 under hyperthermic and hypothermic stress in human hepatoma HepG2 cells. Western blot analysis indicates that there was a statistically significant increase of Hsp70 expression under thermal stresses. Immunohistochemically, the distribution of inducible Hsp70 in stressed cells showed a granular pattern mostly in the cytoplasm. At subcellular level, Hsp70 was localized in the nucleus, vacuoles, cytoskeletal components and dispersed throughout the cytoplasm. Accumulation of Hsp70 in cells under hypothermia could be related to restitution of cell equilibrium modified by this thermal stress condition. The protective effect of hypothermia could be associated with promotion of Hsp expression. We suggest that hypothermia is a stress capable of inducing Hsp70 expression in human HepG2 cells.     

Somatic plasticity of neural stem cells: Fact or fancy?

Several studies have described the potential for embryonic and adult neural stem cells to differentiate into non-neural cells such as muscle and blood, tissues that are derived from non-neuroectodermal germ layers. This raised the exciting possibility that these cells possessed a broader range of differentiation potential than originally thought and raised interesting prospects for possible transplantation utilization. However, a number of recent reports have raised questions about whether the phenomena observed actually represented true somatic plasticity. In this review, we critically analyze these studies with the aim of providing some criteria by which future studies that address this important problem may be evaluated.     

Embryonic stem cells: are useful in clinic treatments?

It is not uncommon to find statements in the social media and even in some scientific journals declaring that embryonic stem cells can be used in human medicine for therapeutic purposes. In our opinion, this statement does not fit the medical reality. To go into this subject in depth, and if possible to clarify it, we reviewed the most recent literature on clinical trials conducted with embryonic stem cells, concluding that up to the present time, there is only one ongoing clinical trial being carried out with these types of cells to treat a small group of patients with spinal cord injury. The results of this trial have still not been published. In conclusion, at present, there is only evidence of one phase I clinical trial conducted with embryonic stem cells, in comparison to the numerous trials conducted with adult stem cells.     

Expression of the Thomsen-Friedenreich antigen and of its putative carrier protein mucin 1 in the human placenta and in trophoblast cells in vitro

The Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope Galbeta1-3GalNAcalpha-O-) has been known for a long time as a carcinoma-associated antigen. In normal tissues the occurrence of TF antigen is restricted to a few immunologically privileged areas. Here we report on the identification of the TF epitope and its putative carrier protein mucin 1 (MUC1) in human placental tissue, on isolated trophoblast cells in vitro and on trophoblast tumour cell lines BeWo and Jeg3. Cryosections of placental and decidual tissues of the first, second and third trimester were double stained with monoclonal antibodies directed against the TF epitope (IgM) and against MUC1 (IgG). In the first trimester of pregnancy we found strong expression of TF antigen and MUC1 at the apical side of the syncytiotrophoblast directed towards the maternal blood. This expression was consistent in the second trimester of pregnancy, and to a lesser degree in the third trimester. In addition, we found positive staining for TF antigen and MUC1 on extravillous trophoblast cells in the decidua during the first and second trimester of pregnancy. Trophoblast tumour cells of the cell line BeWo, which form a syncytium in vitro, were also positive for TF antigen and MUC1, whereas Jeg3 cells, which are unable to form a syncytium, expressed only MUC1. Freshly isolated trophoblast cells from first trimester placentas showed strong staining for MUC1; however, only a few of these cells (less than 1%) were positive for TF antigen, and might consist of digested fragments of the syncytium. In summary, TF antigen and MUC1 are expressed by the syncytiotrophoblast at the feto-maternal interface and by extravillous trophoblast cells invading the decidua, whereas villous cytotrophoblast cells in situ as well as freshly isolated trophoblast cells from first trimester placentas only express MUC1 but not TF antigen.     

Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.     

Differences in the products of mitochondrial protein synthesis in vivo in human and mouse cells and their potential use as markers for the mitochondrial genome in human–mouse cell hybrids

The proteins synthesized in the mitochondria of mouse and human cells grown in tissue culture were examined by electrophoresis in polyacrylamide gels. The proteins were labelled by incubating the cells in the presence of [(35)S]methionine and an inhibitor of cytoplasmic protein synthesis (emetine or cycloheximide). A detailed comparison between the labelled products of mouse and human mitochondrial protein synthesis was made possible by developing radioautograms after exposure to slab-electrophoresis gels. Patterns obtained for different cell types of the same species were extremely similar, whereas reproducible differences were observed on comparison of the profiles obtained for mouse and human cells. Four human-mouse somatic cell hybrids were examined, and in each one only components corresponding to mouse mitochondrially synthesized proteins were detected.