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Challenges and Opportunities in Stem Cells

Challenges and Opportunities in Stem Cells

Christian Elmshaeuser, Ina Zoeller, Darisuren Anhlan, Ewald Beck, Bruno Peault, Olivier Tabary, and Una Chen

Abstract

Background-Purpose of this study: Tissue-specific stem cell lines are useful tools for cell biology studies. Information on respiratory tissue cell lines is limited. A doxycycline-regulated epithelial precursor cell line was established from the lung tissue of a tTAxSV40 Tag double transgenic mouse. In this study, we have characterized this cell line in vitro & in vivo, and found to mimic a rare subpopulation of club- and pneumocyte type II-dual cells.

Methods: It was partially characterized using cell viability and death assays, H3-thymidine incorporation assay, chloride efflux assay, Western blotting of proteins secreted, RT-PCR assays for RNA isolated. In addition, immunedeficient SCID mice were used as hosts for implantation of this precursor cell line, and feed with/without doxycycline containing water. Immunofluorescent typing using different antibodies were used to characterize the implanted lung.

Results: This cell line was found to mimic a rare subpopulation of club- and pneumocyte type II- dual cells with multiple phenotypes. Cell growth was doxycycline-regulated and observed only when doxycycline was omitted from the medium or present at concentrations up to 1 µg/ml, higher concentrations were inhibitory. ACT+ ciliated cells were found upon implantation into immune-deficient mice, in addition. Cell growth was doxycycline-regulated in vitro. When transplanted subcutaneously into immunedeficient mice, these cells migrated to the lung to form organized chimeric structures of donor and host origins, with club cells in the terminal bronchioles, ACTciliated cells along the epithelial lining, and pneumocyte type II-cells in the alveolar interstices. No such homing of donor cells to the lung was observed when the implanted mice were fed doxycycline-containing water.

Discussions-Conclusions: This lung stem cell line might be able to provide us with an insight into the differentiation pathway of lung epithelial cells as well as with some understanding of the nature of air trophic-pulmonary epithelial cells. The results of this study underline the possibility of a future application for somatic (stem / precursor) cells in tissue replacement and tissue engineering of the damaged lung. Its ability to secrete and deliver soluble protein, might be a potential novel way for drug delivery. In addition, stem cells are thought to proliferate and differentiate in response to a deficiency or as a result of injury. Successful migration to the target organ and subsequent maturation of these precursors could be attributed to a requirement of lung stem cells to search for an aerated environment. Our findings challenge some current concepts of stem cell biology.This lung stem cell line may become a rich source of cells for tissue engineering and cell-based therapy for lung injury. The route and protocol established for cell introduction into the lung may provide a novel alternative to delivery of soluble protein substances through the airways. This lung stem cell line might also be modified to provide models for screening drugs against respiratory infection.

Richard L. Momparler, Sylvie Côté, and Louise F. Momparler

Abstract

Complete eradication of leukemic stem cells (LSCs) in patients with acute myeloid leukemia (AML) is required for curative therapy. Epigenetic alterations that involve gene-silencing by DNA methylation by DNMT1, methylation of H3K27 by EZH2 histone methyltransferase (HMT) and methylation of H3K9 by G9a HMT may play a major role in the development of AML. The major action of these epigenetic alterations is the silencing of the genes that program differentiation of AML cells. Inhibitors of DNA and histone methylation have the potential to reverse this block in differentiation. If tumor suppressor genes (TSGs) contain two gene-silencing markers, such as DNA methylation and H3K27me3, they may not be fully reactivated with only an inhibitor of DNA methylation, such as 5-aza-2’-deoxycytidine (5-AZA-CdR), but may also require an inhibitor of EZH2. In support of this model is the synergistic antileukemic action as shown by a colony assay on AML cells using 5-AZA-CdR in combination with 3-deazaneplanocin A (DZNep), a potent inhibitor of EZH2. A similar type of interaction can occur when TSGs are silenced by DNA methylation and the G9a methylation of H3K9me2, a second gene-silencing marker. Treatment of these AML cells with 5-AZA-CdR and BIX01294, an inhibitor of G9a, also results in a synergistic antileukemic action. Leukemic cells that contain 3 different gene-silencing markers: DNA methylation, H3K27me3 and H3K9me2 may require 3 different inhibitors for maximal antineoplastic activity. This result was observed when the AML cells were treated in with 5-AZA-CdR, DZNep and BIX01294. The aim of this study was to demonstrate that epigenetic agents that target DNA and histone methylation have remarkable antineoplastic activity against myeloid leukemia cells. The second aim was to propose a dose-schedule for these epigenetic agents that can be evaluated in a clinical trial in patients with advanced AML for its potential to eradicate LSCs. One of the most sensitive targets for chemotherapeutic intervention in LSCs is the block in differentiation due to gene-silencing by DNA and histone methylation. Epigenetic agents that have the potential to reverse this block merit clinical investigation with high priority.

Nitin T. Telang

Abstract

Chemo-resistant cancer stem cells represent a sub-population of cancer initiating phenotype in primary cancer. These cells evolve in to a metastatic phenotype via activation of multiple cell signaling pathways for cancer cell survival and epithelial-mesenchymal transition. Reliable cancer stem cell models represent a valuable experimental approach for drug discovery platform to identify efficacious testable alternatives against therapy-resistant cancer. Present commentary provides a systematic discussion of relevant conceptual and technical aspects of cancer stem cell biology and its significance for therapeutic alternatives.     

Priya Hays

Abstract

Cancer stems cells are cells in tumors that have self-renewing capabilities and proliferation, and are partly responsible for tumor growth, metastasis and drug resistance, and have been associated with multidrug resistance and epithelial-mesenchymal transition. mRNA stemness index or mRNAsi is a machine learning tool that uses the application of algorithms to find associations between cancer stemness and tumor prognostic signatures. mRNAsi predicts gene mutation status and identifies tumor signaling pathways. Clinical tier grading is a common feature for stratifying the presenting features and symptoms of patients in several diseases. This study is a review article that summarizes studies in lung cancer, gastric cancer, hepatocellular carcinoma and glioblastoma that use mRNA stemness index machine learning tools to identify differentially expressed genes, characterize the tumor microenvironment and tumor mutational burden, and determine clinical endpoints. A prognostic signature is shown in this paper as determined by mRNAsi high and low values, and a clinical tier grading system is proposed that categorizes cancer stemness presenting characteristics. This clinical grading tier system demonstrates a relationship between cancer stemness and immune checkpoint inhibitor efficacy. This type of tiered system for cancer patients and the accompanying workflow proposed may prove useful to oncologists, and has not been performed before, and is unique in the literature.

M. Rucci, F. Onorato, M. Formica, amd R. Ferracini

Abstract

Osteoarthritis (OA) is a widespread degenerative joint disease. The knee arthroplasty has represented for about 40 years the main treatment option, when pain and functional decrease became unbearable for the patient. The knee arthroplasty represents a challenging surgical operation, in which, based on current data available, about 20% of the patients are left totally or partially unsatisfied with the clinical result.

A recent therapeutic alternative for mild-to-moderate grade OA (Kellgren-Lawrence grade II to III) in order to delay (or ultimately avoid) the need for prosthetic surgery, is a treatment with Adipose-Derived Stem Cells (or ADSCs). The safety and efficacy in terms of functional improvement, cartilage repair and inflammation reduction were already reported for OA treatment through a single intra-articular injection of Micro-Fragmented Autologous Fat Tissue (M-FAT), which is known to be rich in ADSCs. This efficacy period appears to be ranging from several months to a few years.

Research has shown that the effect of single-dose M-FAT treatment, despite being evident in most cases, is not statistically uniform among the studies now available. This is also due to different treatment protocols, that leave to every research group the ultimate choice of preparation methods, rules and exclusion criteria.

It is possible, because of this, to hypothesize a new kind of treatment protocol based on a single surgical harvesting operation. The extract would then need to be cryopreserved and administered multiple times over a several-years period. We hope, thanks to the subsequent injections of purified extract, to prolong and boost the effect of the treatment.

We tried to elaborate, based on our experience in the field, a new protocol for this multi-injection treatment. This protocol is going to be applied to a small number of patients and evaluated over time, to elaborate the actual benefit from a treatment like this.

Kerry Thompson, Michelle Kanemori, Emily Kimes, Evthokia Hobbs, Jennifer Phan, Travis Wilson, Alex Wertheimer, Taylor Yoshida, Diana Flores-Barnett, Minji Kang, and Cameron Auyang

Abstract

Cell-based therapy is likely to become a clinically useful tool to treat severe neurological disease. Disease states such as intractable temporal lobe epilepsy will be candidates for this type of therapeutic approach. Embryonic stem cells are pluripotent, and are considered a viable source for cell replacement strategies. Embryonic stem cells can be genetically manipulated to express reporter molecules, and they can be forced into neural differentiation by controlling the expression of key transcription factors. One strategy for the treatment of temporal lobe epilepsy is to transplant enriched populations of transplantable GABAergic neurons into the damaged, epileptogenic, hippocampus. The goal would be to suppress seizures, and to replace and repair damaged circuits. We genetically engineered an embryonic stem cell -derived neurogenic, fusionogenic cell line with GAD65 and transplanted them into an animal model of temporal lobe epilepsy. We found some evidence of seizure suppression, and also evidence of widespread dispersion from the transplantation site. The pattern of staining of embryonic stem cell reporter molecule that was detected, suggested a pathotropism, and selective involvement of reportedly fusionogenic cell populations within the host. We feel that the evidence suggests that embryonic stem cell-derived neural cells might have a significant capacity to fuse with host neurons, which can cloud the interpretation of cellular replacement and circuit repair. This study, and other investigations that have reported stem cell fusion events in the transplanted host, reveal embryonic stem cell capacities that may not be fully appreciated. More research is needed to fully reveal the potential of cellular therapies using stem cells.