Usefulness of mesenchymal cell lines for bone and cartilage regeneration research

[Abstract] The unavailability of sufficient numbers of human primary cells is a major roadblock for in vitro repair of bone and/or cartilage, and for performing disease modelling experiments. Immortalized mesenchymal stromal cells (iMSCs) may be employed as a research tool for avoiding these problems. The purpose of this review was to revise the available literature on the characteristics of the iMSC lines, paying special attention to the maintenance of the phenotype of the primary cells from which they were derived, and whether they are effectively useful for in vitro disease modeling and cell therapy purposes. This review was performed by searching on Web of Science, Scopus, and PubMed databases from 1 January 2015 to 30 September 2019. The keywords used were ALL = (mesenchymal AND (“cell line” OR immortal*) AND (cartilage OR chondrogenesis OR bone OR osteogenesis) AND human). Only original research studies in which a human iMSC line was employed for osteogenesis or chondrogenesis experiments were included. After describing the success of the immortalization protocol, we focused on the iMSCs maintenance of the parental phenotype and multipotency. According to the literature revised, it seems that the maintenance of these characteristics is not guaranteed by immortalization, and that careful selection and validation of clones with particular characteristics is necessary for taking advantage of the full potential of iMSC to be employed in bone and cartilage-related research.Xunta de Galicia; R2016/036Deputación da Coruña; BINV-CS/2016Xunta de Galicia; R2014/050Xunta de Galicia; CN2012/142Xunta de Galicia; GPC2014/04

Cell Reprogramming, IPS Limitations, and Overcoming Strategies in Dental Bioengineering

The procurement of induced pluripotent stem cells, or IPS cells, from adult differentiated animal cells has the potential to revolutionize future medicine, where reprogrammed IPS cells may be used to repair disease-affected tissues on demand. The potential of IPS cell technology is tremendous, but it will be essential to improve the methodologies for IPS cell generation and to precisely evaluate each clone and subclone of IPS cells for their safety and efficacy. Additionally, the current state of knowledge on IPS cells advises that research on their regenerative properties is carried out in appropriate tissue and organ systems that permit a safe assessment of the long-term behavior of these reprogrammed cells. In the present paper, we discuss the mechanisms of cell reprogramming, current technical limitations of IPS cells for their use in human tissue engineering, and possibilities to overcome them in the particular case of dental regeneration

Generation of Mesenchymal Cell Lines Derived from Aged Donors

[Abstract] Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering.This research was carried out thanks to the funding from Rede Galega de Terapia Celular 2016 (R2016/036) and Grupos con Potencial de Crecemento 2020 (ED431B 2020/55) from Xunta de Galicia, Proyectos de Investigación 2017 (PI17/02197) from Instituto de Salud Carlos III and the Biomedical Research Network Center (CIBER). The Biomedical Research Network Center (CIBER) is an initiative from Instituto de Salud Carlos III (ISCIII). MPR and SRF were granted a predoctoral fellowship from Xunta de Galicia and European Union (European Social Fund)Xunta de Galicia; R2016/036Xunta de Galicia; ED431B 2020/5

Generation and Characterization of Mesenchymal Cell Lines for Osteorchondral Regeneration Research

Programa Oficial de Doutoramento en Ciencias da Saúde. 5007V01[Resumo] A rexeneración do óso e da cartilaxe tras sufrir un traumatismo ou unha enfermidade dexenerativa segue sendo un gran desafío clínico. Debido á súa capacidade de auto-renovación e multi-diferenciación, as células mesenquimais estromais (MSC) son unha fonte celular moi prometedora para a rexeneración destes tecidos, pero a investigación neste campo está limitada pola tendencia das MSC á senescencia ao seren expandidas en cultivo. A inmortalización das MSC permítelles superar a senescencia, o que supón un impulso para os avances na investigación. Neste estudo desenvolveuse un método para inmortalizar MSC derivadas de doantes de idade avanzada mediante inoculación centrífuga de dous xenes de inmortalización: o antíxeno T grande do virus de simio 40 (SV40LT) e a transcriptase reversa da telomerase humana (hTERT). As MSC inmortalizadas son fenotipicamente similares ás MSC primarias e son capaces de diferenciarse cara ás tres liñaxes esqueléticas, aínda que se inclinan cara á ruta de diferenciación osteoxénica. Os condrocitos articulares e os sinoviocitos pódense inmortalizar empregando o mesmo método, pero os condrocitos inmortalizados son metabolicamente diferentes dos condrocitos articulares primarios. Estas células poden ser útiles como parte de modelos in vitro de rexeneración dos tecidos articulares ou de enfermidade osteocondral.[Resumen] La regeneración del hueso y el cartílago tras sufrir un traumatismo o una enfermedad degenerativa continúa siendo un gran desafío clínico. Debido a su capacidad de auto-renovación y multi-diferenciación, las células mesenquimales estromales (MSC) son una fuente celular prometedora para la regeneración de estos tejidos, pero la investigación en este campo se ve limitada por la tendencia de las MSC a la senescencia en cultivo. La inmortalización de las MSC les permite superar la senescencia, impulsando así los avances en la investigación. En este estudio, se ha desarrollado un método para inmortalizar MSC derivadas de donantes de edad avanzada mediante inoculación centrífuga de dos genes de inmortalización: el antígeno T grande del virus de simio 40 (SV40LT) y la transcriptasa reversa de la telomerasa humana (hTERT). Las MSC inmortalizadas son fenotípicamente similares a las MSC primarias y son capaces de diferenciarse hacia los tres linajes esqueléticos, aunque tienen tendencia a seguir la ruta de diferenciación osteogénica. Los condrocitos articulares y los sinoviocitos se pueden inmortalizar utilizando el mismo método, pero los condrocitos inmortalizados son metabólicamente diferentes de los condrocitos articulares primarios. Estas células pueden ser útiles como parte de modelos in vitro de regeneración de los tejidos articulares o de enfermedad osteocondral.[Abstract] Regeneration of bone and cartilage after trauma or age-related degenerative diseases remains a major clinical challenge. Due to their self-renewal and multi-differentiation potential, mesenchymal stromal cells (MSCs) are a promising cell source for bone and cartilage regeneration, but research on this field is impaired by MSCs’ predisposition to senescence when culture-expanded. Immortalization of MSCs allows them to bypass senescence, thus boosting the advances in MSC research. In this study, a method has been developed to immortalize MSCs derived from elderly donors by spinoculation of two immortalization genes: simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Immortalized MSCs are phenotypically similar to primary MSCs and are able to differentiate to the three skeletal lineages, although their multi-differentiation potential is unbalanced towards the osteogenic pathway. Articular chondrocytes and synoviocytes can also be immortalized by the same method, but immortalized chondrocytes are metabolically different from primary articular chondrocytes. These immortalized cells can be useful as part of in vitro models of osteochondral regeneration and disease

Genetic Analysis of Pituitary Thyrotrope Development

The pituitary gland produces polypeptide hormones that regulate many functions including growth, lactation, reproduction, metabolism, and the stress response. Pituitary thyrotrope cells produce the heterodimeric glycoprotein hormone thyrotropin, which is critical for stimulating thyroid gland development and production of thyroid hormone. Less is known about the drivers of thyrotrope cell fate than the other specialized cells in this organ. The transcription factor POU1F1 is critical for generation of thyrotropes, somatotropes and lactotropes, and GATA2 is critical for both thyrotropes and gonadotropes. Additional factors are likely involved in driving thyrotrope fate. SV40-immortalized cell lines have been invaluable for studying the regulation of pituitary hormone production. Here I use two established immortalized cell lines to identify epigenomic and gene expression changes that are associated with adoption of the thyrotrope fate. GHF-T1 cells represent a POU1F1-expressing progenitor which does not produce hormones, and TaT1 cells represent a thyrotrope-like line that expresses POU1F1, GATA2 and thyrotropin (TSH). I also developed a novel, genetically engineered mouse line that expresses SV40 in response to cre recombinase, and I used this line to develop novel pituitary cell lines. These cell lines can be used for transcriptome and epigenome studies to understand the development and function of the pituitary gland. I identified the transcription factors and epigenomic changes in chromatin that are associated with thyrotrope differentiation. I generated and integrated genome-wide information about DNA accessibility, histone modifications, POU1F1 binding and RNA expression data to identify regulatory elements and candidate transcriptional regulators. I identified POU1F1 binding sites that are unique to each cell line. POU1F1 binding sites are commonly associated with bZIP factor motifs in GHF-T1 cells and Helix-Turn-Helix or basic Helix-Loop-Helix motifs in TαT1 cells, suggesting classes of transcription factors that may recruit POU1F1 to unique sites. I validated enhancer function of novel elements we mapped near Cga, Pitx1, Gata2, and Tshb by transfection in TαT1 cells. Finally, I confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice and demonstrated that GATA2 enhances Tshb expression via this element. These data extend the ENCODE analysis to an organ that is critical for growth and metabolism. This information could be valuable for understanding pituitary development and disease pathogenesis. Targeted oncogenesis is the process of driving tumor formation by engineering transgenic mice that express an oncogene under the control of a cell-type specific promoter. Using CRISPR/Cas9 we inserted a cassette with coding sequences for SV40 T antigens and IRES-GFP into the Rosa26 locus, downstream from a stop sequence flanked by loxP sites: Rosa26 LSL-SV40-GFP . These mice were mated with previously established Prop1-cre and Tshb-cre transgenic lines. The majority of Rosa26 LSL-SV40-GFP/+ ; Prop1-cre and all Rosa26 LSL-SV40-GFP/+ ; Tshb-cre mice developed dwarfism and large tumors by 4 weeks. Prop1-cre-mediated activation of SV40 expression affected cell specification, reducing thyrotrope differentiation and increasing gonadotrope cell fate selection. GFP-positive cells from flow-sorted Rosa26 LSL-SV40GFP/+ LSL-SV40-GFP/+; Prop1-cre and Rosa26 ; Tshb-cre mice express PROP1 and TSH, respectively. Tumors from both of these mouse lines were adapted to growth in cell culture. I established a progenitor-like cell line (PIT-P1) that expresses Sox2 and Pitx1, and a thyrotrope-like cell line (PIT-T1) that expresses Cga and Pou1f1. These studies demonstrate the utility of the novel, Rosa26 LSL-SV40-GFP mouse line for targeted oncogenesis and development of cell lines.PHDGenetics and Genomics PhDUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/162910/1/azdaly_1.pd

Die Evaluation von Seneszenzmarkern in der Kultur von dentalen Follikelvorläuferzellen

Für die moderne Zahnmedizin stellen adulte dentale Stammzellen wie dentale Follikelvorläuferzellen (DFVs) vielversprechende zukünftige Behandlungs-alternativen dar, beispielsweise für die Regeneration parodontalen Gewebes. Dafür werden große Mengen von Zellen benötigt, weshalb sie in vitro kultiviert werden müssen. Seneszenz ist dabei ein limitierender Faktor, da sie zu begrenzter Lebenserwartung, Einschränkung der Differenzierungsfähigkeit und verringertem Proliferationspotential führt. Bei DFVs sind Seneszenzmarker bisher noch nicht genau untersucht. Ziel der Arbeit war die Analyse von Seneszenzmarkern, um zu prüfen ob DFVs in hohen Passagen seneszent werden. Dazu wurden Zellmorphologie, Histologie, Telomerlänge, die Expression von Telomerase-assoziiertem TEP1 und osteogene Differenzierung untersucht. Die vorliegende Arbeit liefert neue Erkenntnisse zur Seneszenz von langzeit-kultivierten (bis Passage 18) dentalen Follikelvorläuferzellen. P14 scheint eine kritische Phase bei der Ausbildung seneszenter Tendenzen zu sein. So besitzen DFVs eine veränderte Zellmorphologie, exprimieren seneszenz-assoziierte β-Galaktosidase und zeigen vermindertes osteogenes Differenzierungspotential, sowie reduzierte Expression von TEP1. Telomerverkürzungen sind jedoch tendenziell. Weiterhin lässt sich nach Stammzellmarker-Analyse die Hypothese aufstellen, dass CD146 eine Rolle bei der Entstehung von seneszenten DFVs spielt. DFVs zeigen bis P18 keine stark ausgeprägte, zellzyklushemmende Seneszenz. Der Fokus zukünftiger Studien sollte daher auf Passagen größer 18 liegen. Ebenso sind weiterführende Studien nötig, welche die Bedeutung von CD146 für die Entstehung von Seneszenz untersuchen

Regulation and patterning of cell differentiation and pluripotency

The development of a multicellular organism from an embryo is one of the nature's most remarkable phenomena. Deciphering how this transformation occurs is a fundamental challenge in biology with profound biomedical implications. Insights into the molecular signals guiding developmental patterning may provide design strategies to promote multicellular structure formation in applications such as tissue engineering and regenerative medicine. In this thesis, we explored the applications of controllable gene expression techniques in combination with engineering strategies in regulation and patterning of cell differentiation and pluripotency, by pursuing three related research projects: 1. Reversible immortalization of cardiomyocytes to enable their proliferation necessary for obtaining large cell numbers 2. Patterning of the delivery of Doxycycline (Dox), the expression modulator of inducible BMP-2 expression vector, to mesenchymal stem cells cultured in a microfluidic 3. Patterning of the Nanog gene expression in embryonic stem cells, using a microfluidic device, to establish differentiation - pluripotency boundaries that mimic the developmental processes in vivo. In the first project, we developed a novel strategy for controlled expansion of non-proliferating primary neonatal rat cardiomyocytes by lentivector-mediated cell immortalization, and then the reversal of the phenotype of expanded cells back to the cardiomyocytes state. Primary rat cardiomyocytes were transduced with simian virus 40 large T antigen (TAg), or with Bmi-1 followed by the human telomerase reverse transcriptase (hTERT) gene; the cells were expanded; and the transduced genes were removed by adenoviral vector expressing Cre recombinase. The TAg gene was more efficient in cell transduction than the Bmi-1/hTERT gene, based on the rate of cell proliferation. Immortalized cells exhibited the morphological features of dedifferentiation (increased vimentin expression, and reduced expression of troponin I and Nkx2.5) along with the continued expression of cardiac markers (α-actin, connexin-43, and calcium transients). After the immortalization was reversed, cells returned to their differentiated state, as evidenced by molecular and functional properties inherent to terminally differentiated cardiomyocytes. This strategy for controlled expansion of primary cardiomyocytes by reversible gene transfer could provide large amounts of a patient's own cardiomyocytes for cell therapy, and enable controlled in vitro study of cardiogenesis. In the second project, we developed a novel patterning strategy by using inducible gene expression systems in conjunction with simple multi-laminar fluidic techniques, which can directly pattern the expression of particular gene at transcriptional level. Using osteogenic differentiation of human mesenchymal stem cells as a model, we describe a novel approach to spatially regulate the expression and secretion of bone morphogenetic protein (BMP-2) in a two-dimensional field of cultured cells, by flow patterning the modulators of inducible BMP-2 gene expression. We first demonstrated a control of gene expression, and control of osteogenic differentiation of the cell line with inducible expression of BMP-2. Then we designed laminar flow systems, with patterned delivery of Doxycycline (Dox), the expression modulator of inducible BMP-2 expression vector. The patterned concentration profiles were verified by computational simulation and dye separation experiments. Experiments conducted in the flow systems for a period of three weeks showed the Dox concentration dependent osteogenic differentiation, as evidenced by mineral deposition. This strategy combining inducible gene expression with laminar flow technologies provides an innovative way to engineer tissue interfaces. In the third project, we further developed the patterning strategy for gene expression to form boundaries of different gene expression domains in cultures of mouse embryonic stem cells. Using Nanog safeguarded embryonic pluripotency as a model; we demonstrated controlled Nanog expression, which lead to controlled early differentiation under the exposure or withdrawal of varied small molecules, as evidenced by alkaline phosphatase (AP) staining, immunofluorescent staining, and gene expression analysis. By patterning Nanog gene expression, as well as soluble factors in the laminar fluidic system, we successfully developed varied differentiation - pluripotency boundaries between Nanog expressing pluripotency zones and Nanog suppressed early differentiation zones from the same population of cells, which mimic the development process in vivo. Mechanistic insights can be gained on dissecting the signaling pathways that drive multicellular patterning during the natural processes of embryonic and adult development. In summary, we demonstrated that controlled expansion of non-proliferating primary cells can achieved by reversible genetic manipulation, and that varied continuous, graded pluripotency - differentiation boundaries can established by patterning the expression of target genes via a simple laminar fluidic system. Taken together, these approaches provide innovative models to modulate cell function at the transcriptional level. Additional cooperative research was conducted during my graduate training. The manuscript of this study "Micropatterned Mammalian Cells Exhibit Phenotype-Specific Left-Right Asymmetry" was submitted to Proc Natl Acad Sci U S A., and it is currently under review. We attached this manuscript in appendix

Basal Cell Carcinoma

Basal cell carcinoma is the commonest cutaneous malignancy. The last decade has witnessed exponential research which has broadened our understanding of the pathogenesis of basal cell carcinomas. This is also important from a therapeutic point of view as targeted approach to therapy is now being increasingly experimented. Although it is impossible to condense and present all good research in one book, the authors have to be commended on presenting their research on several aspects of basal cell carcinoma in a succinct manner, which shall not only enhance our understanding of, but also hopefully via this open exchange of ideas pave ways for successful targeted therapy of the commonest human cancer

Ocular Tissue Engineering

Tissue engineering emerged back in the 1990s as a new concept to overcome the problem of tissue and organ failure. Over recent decades, there has been incredible progress towards the regeneration of tissues such as bone, heart valves, cartilage, cornea, and retina. In terms of ocular tissue engineering, despite the scientific and strategic incentive for reconstructing ocular tissues, there is also a tremendous need for novel therapeutic options in treating numerous eye diseases related to tissue failure. The aim of this Special Issue is to discuss tissue engineering applications of ocular tissues including but not limited to cornea, retina, and lenses

MYC transcriptional functions controlling epidermal stem cell self-renewal and differentiation

The oncoprotein MYC has long been recognized as an important stem cell regulator, yet its direct biological contributions have been difficult to determine. MYC activation can induce pleiotropic phenotypes and mediates cellular functions as opposing as cell growth and proliferation, metabolism, differentiation and apoptosis. In addition, functional redundancy with MYCL and MYCN proteins as well as dose dependency, complicates the identification of the most relevant biological functions. Studies in tissues with high proliferative capacity and rapid turnover have shown that MYC is a key regulator of homeostasis by balancing stem cell self-renewal, proliferation and differentiation processes. In skin, MYC induces the exit of epidermal stem cells from their niche, increases proliferation of progenitor cells and subsequently stimulates lineage specific differentiation into interfollicular epidermis and sebaceous glands; yet the direct transcriptional roles of MYC in these processes remained elusive. To gain insight into the transcriptional roles of MYC in epidermal stem cell homeostasis, I performed chromatin immunoprecipitation on microarrays (ChIP-on-Chip) using mouse proximal promoter arrays combined with mRNA expression data that was generated using epidermal cells from wild-type and transgenic K14MycER mice, treated in a time-course from zero to six days with tamoxifen, to induce the ‘Myc’ transgene expression in the basal undifferentiated layers of the epidermis. Data analysis revealed that 2187 genes, which corresponds to 15% of the promoter regions covered, were directly regulated by MYC. To identify genes uniquely regulated by MYC in skin, I performed gene expression studies on mouse skin in which MYC was conditionally deleted in the basal layer of the epidermis. Remarkably, I found that 45% of all repressed genes were related to epidermal maintenance and differentiation. To better understand the mechanism of how MYC induces keratinocytes to differentiate specifically into lineages of sebaceous glands and interfollicular epidermis, I analyzed whether MYC might have directly regulated genes involved in skin differentiation. Here, I focused my studies on a single 2.2 Mb locus located on mouse chromosome 3 designated as the epidermal differentiation complex (EDC). To assess how activation of MYC could influence the expression of genes localized to the EDC, I performed ChIP-on-Chip for MYC, H3K4me3, H3K27me3, as well as transcription factors, which have been described to regulate terminal differentiation in skin, such as CEBPα, OVOL-1, KLF4, TCFAP2-γ and SIN3A, among others. I demonstrated that MYC recruits a specific set of tissue-specific transcription factors to the EDC, (e.g. KLF4 and OVOL-1) and thereby prevents binding of a different and distinct set of genomic regulators, (e.g. CEBPα , MXI1 and SIN3A). Using a combination of mouse models and systems biology tools, I then identified SIN3A as a key regulator in this MYC-dependent transcriptional network. I found that MYC and SIN3A form a negative feedback loop, which is required to balance proliferation and differentiation in epidermis, and both factors are essential to maintain skin homeostasis