Finally, to demonstrate that some cancer methylated genes are also frequently methylated in somatic progenitor stemcells and that their methylation is important for lineage specification, we considered two genes: RUNX3 and AIM2. We selected RUNX3 because, in agreement with previously published data , our methylation arrays showed that, relative to CD34+ cells, RUNX3 was hypomethylated in peripheral lymphocytes but not in peripheral neutrophils. AIM2 was selected because it was previously thought to be a TSG that is frequently hypermethylated in cancer (Figure S8)  and because, unlike RUNX3, it becomes unmethylated specifically in the myeloid lineage (Figure 4B,C). The bisulfite sequencing data confirmed the results obtained with the arrays, showing that the CD34+ cells and the peripheral lymphocytes were densely methylated at the promoter of AIM2 gene whilst the peripheral neutrophils were almost unmethylated (Figure 4B). To determine the role of Figure 2. Promoter DNA hypermethylation and repression of MGMT in hESCs. (A) Bisulfite genomic sequencing of multiple clones of the MGMT promoter in hESCs (I3, H14), normal primary tissues (Pool lymphocytes, normal breast) and two CCLs of lymphoid and breast origin (U937 and MDA-MB-231, respectively). Black, methylated CpG; white, unmethylated CpG; red, CpG not present. The green bar above the diagram of the MGMT CpG island indicates the location of the probe used in the methylation arrays. (B) Relationship between MGMT promoter hypermethylation and expression in hESC, normal, and cancer samples. The upper panel shows the relative methylation signal obtained with the methylation arrays and the lower panel the expression levels of MGMT mRNA relative to GAPDH.
Figure 2 Autophagy influences senescence in mesenchymal stemcells. The self-renewal potential of young mesenchymal stemcells (MSCs) is influenced by their autophagy capacity to regulate the good levels of oncogenic factors like p53 and inflammatory signals like senescence-associated secretory phenotype and IGF-1, which produces overexpression of reactive oxygen species in the mitochondria, accumulation of mutations at DNA levels and acidification in the lisosomal apparatus together with an increase of LMNA in the nucleus. When autophagy is downregulated by the pathologic process, young MSCs become old MSCs in an accelerated way, losing their self- renewal capacity. MSC: Mesenchymal stem cell; ROS: Reactive oxygen species.
To date, there have been no reports documenting or comparing membrane PDs in human CSCs and NSCs de- rived from the liver or other tissues. In the only previous paper specifically designed to determine whether differ- ences in PDs exist in cancer and non-cancer stemcells, we described significantly depolarized CSCs derived from the malignant, human Huh-7 and PLC hepatocyte cell lines when compared to NSCs derived from a non-malig- nant, rodent WBF-344 cell line. 5 However, these findings
Our knowledge of the mechanisms via which radiation induces cell death is based on data of cell survival and cell damage after radiation [1-3] and on the consequences that this damage generates at a cellular , tumoral and normal tissue level [5, 6]. In radiation oncology a growing need exists for the development of clinical-decision-support systems based on prediction models of treatment outcome . The models proposed so far to explain the outcome of tumor growth and adverse effects of radiotherapy have led to the current knowledge that not only DNA damage, but also cell signaling processes in off-target cells (bystander and ascopal effects) [8-11] can be crucial to the effect of radiotherapy [12, 13]. New models should combine both predictive and prognostic data factors from clinical, imaging, molecular and other sources to achieve the highest accuracy to predict tumour response and follow-up event rates [13, 14]. Consequently, models which include the bystander effect would seem to be necessary . Indeed, cell-cell communication between sub-lethally damaged cells after radiotherapy and surviving tumor cells leads to a reduction in the remaining and viable cancer cells . Therefore, non-targeted radiation effects might be considered as the response of the tumor [17, 18] and normal tissues [6, 19] to the stress induced by radiation in the target volume . Mesenchymal stemcells (MSC)  have been investigated for the treatment of cancers as they are able to home onto tumors and become incorporated into their stroma. Moreover, MSC homing is increased after radiation therapy . MSCs can both suppress or promote tumor growth [23-25]. The existing information proposes that, as a response to injury, MSCs might have a role in regenerating tissues. This process occurs upon the activation of these MSCs, which become mobilized, activated and secrete factors that enable a cell therapy microenviroment [26, 27] and the molecules secreted by the activated MSCs (MSCs*) may affect a variety of immune cell lineages and establish a powerful therapeutic field [28, 29]. Taking into account both previous reports and our own experience we have designed this study to investigate the following hypothesis:
A new theory about the development of solid tumours is emerging from the idea that solid tumours, like normal adult tissues, contain stemcells (called cancer stemcells) and arise from them. Genetic mutations encoding for proteins involved in critical signalling pathways for stemcells such as BMP, Notch, Hedgehog and Wnt would allow stemcells to undergo uncontrolled proliferation and form tumours. Taking into account that cancer stemcells (CSCs) would represent the real driving force behind tumour growth and that they may be drug resistant, new agents that target the above signalling pathways could be more effective than current anti-solid tumour therapies. In the present paper we will review the molecular basis of the Notch signalling pathway. Additionally, we will pay attention to their role in adult stem cell self-renewal, and cell fate specification and differentiation, and we will also review evidence that supports their implication in cancer.
9 they share some surface markers, both groups are identified as MSC populations with some similitudes and differences, as shown in Table 1. AD-MSCs as all the adult SCs emerge at some point of the development, and as they age, there is a clear loss in pluripotency and tissue restricted differentiation ability. Although plenty of studies show, that mesenchymal lineage has the potential to be stemcells, some of the requirements to formally demonstrate self- renewal and differentiation in vivo were not that conclusive. Due to the use of the same abbreviation —MSC—, the reference to Stem or Stromal cells requires a characterization of their lineage which has involved incongruities in results and in plenty of literature there is not a consensual test to evaluate stemness (Dominici et al., 2006; Phinney & Senseb́e, 2013). Moreover, as Kfoury & Scadden reviewed, the “Mesenchymal Stem Cell nomenclature proved to be problematic when it became obvious that not all plastic adherent stromal cells have comparable self-renewal and in vivo differentiation ability into multiple lineages” (2015). All this controversy led the International Society for Cellular Therapy (ISCT) to resolve the nomenclature clarification: “restricting the use of the term Mesenchymal StemCells for cells that meet the stem cell criteria and recommending the term Multipotent Mesenchymal Stromal Cells for any fibroblast-like plastic adherent cells regardless of tissue of origin” (2015). Briefly after the ISCT added minimal criteria to the identification of real stemcells: CD105+, CD73+, AND CD90+; CD45-, CD34-, CD14- (CD11b), and CD79α- (CD19-), and HLA-DR surface molecules in addition to the ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages in vitro as shown in Table 1 (Dominici et al., 2006). These immunological biomarkers are specific for human cells, as the rodent models present a slightly different pool of CDs (Chamberlain, Fox, Ashton, & Middleton, 2007).
Early evidence for stemcells in the prostate came from a set of experiments involving androgen withdraw- al from both human and rat prostate [12–14]. The existence of stemcells in the prostate is probably best illustrated by animal studies investigating the effect of androgen on the prostate. Castration leads to rapid involution of the gland, but once androgen levels are re- stored, the gland completely regenerates. As this cycle of involution–regeneration can be repeated many times, a population of long- lived, prostatic epithelial stemcells must exist .
certainly should play a role in the metabolic shift that enables somatic reprogramming to stemness because the physiology of mitochondria is inextricably linked to energy metabolism . Specifically, mitochondrial structure and function have been suggested to be indicators of stem cell competence because low mitochondrial activity and relatively under-developed mitochondrial networks have been confirmed to be common features of stemness [43-48]. Vessoni and colleagues  hypothesized that autophagy could play an important role in mediating the remodeling of differentiated cells to a pluripotent state during the generation of iPSCs. Mitophagy would promote mitochondrial degradation during iPSC generation, allowing differentiated cells to reduce the amount of this organelle to ESC-like levels. To test a “metabolic state hypothesis” that links the mitochondrial state and cellular bioenergetics to the state of differentiation, Vessoni and colleagues  suggested that an increase in the number of developed mitochondria and the mitochondrial mass in iPSCs generated from autophagy-deficient cells (ATG7 -/- ) would argue for a pivotal role for autophagy during reprogramming. In the same way, the generation of iPSCs from differentiated cells might also be positively influenced by autophagy modulation. Because mitochondrial fission is a mediator of mitochondrial turnover (i.e., mitochondrial fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy) and because inhibiting mitochondrial fission results in the specific inhibition of mitochondrial autophagy before the phagophore is assembled [50, 51], we recently envisioned that pharmacological perturbation of mitochondrial dynamics before and after iPSC generation may illuminate mitophagy as a pivotal mechanism driving somatic reprogramming to stemness. Our current findings provide new insight into how mitochondria division is integrated into the reprogramming factor-driven transcriptional network that specifies the unique pluripotency of stemcells. Our data strongly suggest for the first time that the occurrence of mitophagy may be involved in the selective turnover of mitochondria prior to and during the reprogramming of somatic cells to iPSCs. In light of recent studies suggesting that changes in metabolism may play a role in enabling the reprogramming process to occur, instead of being a consequence of acquiring a pluripotent state, our data confirm a causal correlation between the bioenergetic state of somatic cells and their reprogramming efficiency. Future studies should elucidate whether the ability of mitophagy to directly shift the oxidative:glycolytic production ratios closer to
Preliminary experience with clinical hepatocyte trans- plantation during the past decade has provided proof of concept that cell therapy can be effective for the treat- ment of some liver diseases. Recent progress in cell bi- ology resulting in the isolation and characterization of hepatic stemcells and progenitor cells further in- creased the expectation for a new approach to the treatment of genetic and chronic liver disease. Several potential sources have been identified of hepatic stem/ progenitor cells exhibiting both differentiation to- wards the hepatic lineage in vitro and hepatic paren- chymal repopulation with liver-specific metabolic ac- tivity in liver-injured animal models. However, a few of these results proved to be poorly reproducible in dif- ferent laboratories, and it was recognized that some initial optimistic conclusions were drawn from incor- rect interpretation of experimental data or from insuf- ficient knowledge of the mechanisms involved in tissue regeneration. Moreover, only modest results have emerged so far from ongoing clinical experience in- volving the use of putative stemcells in liver disease. There is much need for a joined effort to concentrate the resources on a specific cell population, in order to better characterize its function, to assess its safety and
The Hh pathway plays a central role during embryo development to give rise to the distinct structures and organs integrated in a normal individual. This is achieved through the key action of Hh signalling on fate and proliferative capacities of distinct sets of embryonic stemcells. Moreover, in adult individuals Hh also plays important roles control-ling the normal self-renewal and maintenance of tissues as it is able to regulate the proliferative activity of stemcells and their differentiation toward mature cells through pro-genitor or transient-amplifying cells. For instance, in nor-mal adult tissues, expression of several components of the Hh signal transduction machinery has been detected, such as Ptc and Gli in sets of stemcells of the central nervous system and gut epithelium [22–26]. Indeed, increasing evidence indicates that the Hh pathway can be transiently reactivated by tissue damage to stimulate tissue repair, including peripheral nerve regeneration [27–29]. In this way, it has been shown that Hh signalling can regulate cell proliferation through the activation of the cell cycle regulatory proteins cyclin D and E . Although less clear, the control of cell differentiation by Hh signalling might occur through the induction of protein secretion, including neurotrophic and angiogenic factors .
Studies in brain cancer patients and rodent evidences that radiation-related neurofunctional sequelae are associated with a variety of anatomical changes that occur in the irradiated non-tumoral tissue (Makale et al., 2017). Immediately after radiation, brain exhibits vascular damages, oligodendrocyte loss, demyelination and neuroinflammation. Radiation-induced brain injury also disrupts the neurogenic niches located at the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. Moreover, brain injury also affects neuronal dendritic spines and white matter, leading to necrosis of specific areas. The discovery of the negative effects induced by radiation in the non- tumoral tissue has promoted the development of strategies to minimize radiotherapy side effects. In this context, stem cell-based therapy represents a novel alternative to attenuate radiation-induced brain injury (Acharya et al., 2011, 2015; Joo et al., 2012; Piao et al., 2015). In this line, Joo et al. (2012) described the benefits of supplementing whole-brain irradiated mice with fetal mouse neural stemcells (NSCs), which were injected via tail vein 24 h after radiation. The irradiated brain induced homing of the exogenous NSCs, which differentiated along glial and neuronal lineages. Two months after NSC administration, mice showed inhibited radiation-induced hippocampus atrophy and preserved short-term memory. Similarly, human embryonic stem cell-derived oligodendrocyte progenitors (hOPCs) have provided promising results. After bilateral injections into the corpus callosum of rats, hOPCs were able to remyelinate the brain and ameliorate radiation- induced cognitive dysfunction (Piao et al., 2015). However, stem cell-based therapies proposed in current studies present some restrictions that need to be solved if translation to human is sought (Ramos-Zuriga et al., 2012). First, several studies used stemcells with scarce availability and whose isolation procedure is highly invasive (e.g., NSCs). Second, the routes used for administration have limited effectiveness (e.g., systemic transplantation renders reduced concentration of transplanted cells in the brain) or requires invasive techniques that risk host safety (e.g., intracranial injections). Here we explored the non-invasive intranasal delivery of human mesenchymal stemcells (hMSCs) derived from adipose tissue to prevent radiation-induced brain damage in a mouse model of whole- brain radiation. Our results demonstrated that transplanted hMSCs promoted neuroprotection and improved neurological function after irradiation, without compromising survival of glioma-bearing mice.
Cosmeceuticals are a safe and effective way to improve the undesired effects of aging. There are various agents that can be applied to address wrinkles, fine lines, and hyperpigmentation. Cos- meceuticals exert local effects, without systemic absorption. They are also generally well tolerated and no major adverse effects have been observed. Many different options exist, including peptides, growth factors, cytokines, and stemcells, which have joined the cosmeceutical armamentarium.
MatrigelBD is a natural hydrogel scaffold mainly composed of laminin, collagen IV, heparin sulfate proteoglycans, and entanctin/nidogen. Several growth factors, such as TGF-, epidermal growth factor (EGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), neural growth factor (NGF), tissue plasminogen activators, and others are also included in its composition. MatrigelBD is a solubilized basement membrane extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma (Hughes et al., 2010). In preliminary studies, MatrigelBD has been used in cardiomyocyte progenitor transplantation experiments, in a cardiac infarction animal model, and to promote in vivo angiogenesis (Ou et al., 2011; Bhat et al., 2012), and stimulates embryonic stemcells (ESC) to survive and differentiate in vitro (Uemura et al., 2010). One report showed that dental pulp stemcells (DPSC) co-cultured within MatrigelBD maintained their osteogenic
In human medicine, it has been shown that MSCs are a clinical promise for articular cartilage regeneration. Several authors reported studies that demonstrate the effectiveness of MSCs in OA treatment. In relation to ASCs, the first case report was published in 2001 . During the last decade, these cells have attracted great interest because they have been demonstrated to be safe and efficient for articular cartilage regeneration in several trials. In recent years, IA injection of ASCs in knee OA showed clinical, radiological, arthroscopic, and histological evidence at 6-month follow-up . Among other studies, the IA injection of these stemcells (isolated from abdominal subcutaneous fat tissue) in severe knee OA, reported that clinical outcomes (pain, function knee, return to sport) of the low- and medium-dose groups tended to deteriorate after 1 year, while those of the high-dose group tended to plateau after 1 year, until 2 years . Recently, Spasovski et al. (2018) have demonstrated that the use of ASCs from subcutaneous fat in knee OA improves clinical symptoms and reduces pain at 3 months, obtaining the best results at 6 months . ASC therapy in OA has shown chondrogenesis potential, both for the infrapatellar- and suprapatellar-derived ASCs [50,84]. A greater chondrogenesis potential has been reported by infrapatellar ASCs compared to suprapatellar in vitro and in vivo [84,85]. In addition, the suprapatellar-derived ASCs transplantation in a severe knee OA mouse model diminished inflammation and cartilage degenerative grade, increasing the synthesis of glycosaminoglycan and inducing endogenous chondrogenesis . These effects may be due to ASCs-mediated reduction of pro-inflammatory cytokines and chemokines, apoptosis of chondrocytes, hypertrophic and fibrotic chondrocyte phenotypes, and collagenases .
The protein expression profile of MSCs may reveal potential hazards associated with senescence and tumoral transformation that may occur during culture. Proteomic is a valuable tool for human MSC characterization following physiological modifications of the phenotypes of MSCs and identification of possible changes occurring during expansion. Mass spectrometry-based comparative membrane proteomics can enable the identification of novel cancer biomarkers by distinguishing proteins that change membrane localization between normal and malignant tissues and cells. The combination of analyzers and other types of available components has led in recent years to a long list of devices designed specifically for each type of molecule. Specifically, the range of platforms designed for the analysis of peptides and proteins has been adapted specifically to different qualitative and quantitative techniques (Table 1). This review describes proteomic techniques currently applied or prospectively applicable to MSC studies.
Mutations of APC in colorectal carcinoma (CRC) occur mostly in a mutation cluster region that is located approximately in the middle of the coding sequence. The mutations lead to frame shifts or stop codons, resulting in the generation of truncated APC proteins that lack several of the 20-aminoacid repeats that interact with β-catenin and all the SAMP interaction domains for AXIN/conductin. It is thought that the resulting disturbance of the architecture of the β-catenin destruction complex is responsible for the stabilisation of β-catenin. Moreover, truncated APC also lacks several nuclear export sequences (NES) thought to be important for an APC-mediated export of β-catenin from the nucleus. In summary, APC mutations lead to accumulation of β-catenin and constitutive transcription of WNT target genes in the absence of exogenous WNT factors. It is not clear why CRC cells retain the truncated APC versions, i.e., why cancer-generating mutations do not completely abolish APC expression. Interestingly, a special form of attenuated FAP, in which patients develop much fewer polyps than in the classical FAP, is characterised by mutations that generate shorter APC proteins. This indicates that the truncated APC retains some function that is required for robust tumorigenesis. β-Catenin is mutated in up to 10% of all sporadic colon carcinomas by point mutations or in frame deletions of the serine and threonine residues that are phosphorylated by GSK3B. These mutations result in stabilisation of β-catenin and activation of WNT signal-ling. β-Catenin and APC mutations are mutually exclusive, possibly reflecting the fact that both components act on the same pathway .
Figure 3 | Model of bone-marrow HSC niches. Endosteal bone surfaces are lined with stromal cells. Spindle-shaped N-cadherin-expressing osteoblasts (SNOs) serve as niche cells to maintain quiescence and prevent differentiation of attached haematopoietic stemcells (HSCs). The quiescent endosteal niche would maintain dormant HSCs long- term. In response to injury, quiescent HSCs might be activated and recruited to the vascular niche. The self-renewing niche would contain quiescent HSCs intermingled with dividing HSCs. Self-renewing HSCs produce multipotential progenitors (MPPs) either by divisional or environmental asymmetry. More HSCs can be generated by symmetrical divisions which might provide the vascular niche with new HSCs. Whether HSCs long-term self-renew in the vascular niche remains to be determined, and it is probable that influx of HSCs from endosteal niches is necessary to ensure prolonged haematopoietic-cell production at the vascular niche. HSCs in the vascular niche promote differentiation and expansion along megakaryocytic and other myeloid-cell lineages, particularly in response to injury. MPPs can give rise to all haematopoietic lineages, including B-cell precursors attached to randomly distributed CXC-chemokine ligand 12 (CXCL12)-expressing stromal cells that constitute a B-cell niche 82 . Unidentified T-cell
Embryonic stem (ES) cells, however, are derived from the isolated inner cell masses (ICM) of mammalian blastocysts. The continuous in vitro subculture and expansion of an isolated ICM on an embryonic fibroblast feeder layer (human or murine) leads to the development of an embryonic stem cell line. In nature, however, embryonic stemcells are ephemeral and present only in the ICM of blastocysts. The cells of the ICM are destined to differentiate into tissues of the three primordial germ layers (ectoderm, mesoderm and endoderm) and finally form the complete soma of the adult organism. ES cells can be expanded in vitro very easily and, under optimal culture conditions, divide symmetrically to give two daughter cells. ES cell lines express the telomerase gene, the protein product of which ensures that the telomere ends of the chromosomes are retained at each cell division, preventing the cells from undergoing senescence. These cells also retain a normal karyotype after continuous passage in vitro, thus making them truly immortal. The earliest human embryonic stem cell (hESC) lines derived in our laboratory have been maintained continuously in culture for over 300 population doublings, a figure that surpasses the theoretical Hayflick limit of 50 population doublings. 9–11
Retinal and optic nerve diseases are degenerative ocular pathologies which lead to irreversible visual loss. Since the advanced therapies availability, cell-based therapies offer a new all-encompassing approach. Advances in the knowledge of neuroprotection, immunomodulation and regenerative properties of mesenchymal stemcells (MSCs) have been obtained by several preclinical studies of various neurodegenerative diseases. It has provided the opportunity to perform the translation of this knowledge to prospective treatment approaches for clinical practice. Since 2008, several first steps projecting new treatment approaches, have been taken regarding the use of cell therapy in patients with neurodegenerative pathologies of optic nerve and retina. Most of the clinical trials using MSCs are in Ⅰ/Ⅱ phase, recruiting patients or ongoing, and they have as main objective the safety assessment of MSCs using various routes of administration. However, it is important to recognize that, there is still a long way to go to reach clinical trials phase Ⅲ-Ⅳ. Hence, it is necessary to continue preclinical and clinical studies to improve this new therapeutic tool. This paper reviews the latest progress of MSCs in human clinical trials for retinal and Sonia Labrador-Velandia, María Luz Alonso-Alonso, Sara Alvarez-Sanchez, Jorge González-Zamora,
7Ke tKerapeutic bene¿t reported from allogeneic stem cell transplantation is attributed to tKe graft versus tumor effect produced by tKe infused donor immune effector cells to eradicate tumor cells. However, the immune response produced by the donor immune effector cells is not speci¿c creating not only the graft versus tumor effect, but also the detrimental effects of graft versus host disease. Recent reports have shown that the infusion of collected ³bystander´ lymphocytes from the stem cell autograft correlates with lymphocyte recovery and clinical outcomes in patients undergoing autologous stem cell transplantation (ASCT), similar to the graft versus tumor effect seen in the allogeneic stem cell transplantation without the adverse effects of graft versus host disease. The discovery that host immune effector cells collected at the same time as the stemcells can improve clinical outcomes post-ASCT, suggest that autograft can be viewed not only as a therapeutic maneuver to recover bone marrow function after deliver high-dose chemotherapy, but also as an adoptive immunotherapeutic intervention capable of eradicating tumor cells in cancer patients. In this article, we review how to enhance host immune effector cells collection, the different immune effector cells collected and infused from the stem cell autograft, and their association with clinical outcome post-ASCT. Key words: Graft versus tumor effect, stem cell.