Control Stem Cell Fate Research Articles

3081 - Geometrical Control of Stem Cell Fate by Self-Organization of Cytoskeleton

The commitment of mesenchymal stem cells (MSCs) differentiation lineage can be controlled by physical signals, such as geometric cues of extracellular matrix (ECM). It is well known that the physical stimuli drive focal adhesions (FAs) maturation, actin polymerization and stress fiber formation, yet how cell shape controls MSC differentiation through the FA-mediated signals or FA-cytoskeleton organization remains largely unknown. To determine how geometric cues direct MSC lineage commitment, a technique of microcontact printing is performed to shape MSCs. We find that the geometric cues control osteogenic-adipogenic switch in MSC lineage commitment through regulating the organization of FAs, actin filaments, intermediate filaments, microtubules and nucleus, which further influences the distribution of cellular visco-elasticity and traction forces. Our data demonstrate a role for FA signaling in dictating MSC differentiation regulated by geometric cues.

Abstract 2073: Transcriptome profiles of cancer stem-like cells in patient-derived diffuse intrinsic pontine glioma (DIPG)

Abstract Diffuse intrinsic pontine glioma (DIPG) is a rare, but lethal childhood cancer with a 5-year survival less than 1 %. Genetic profiling of DIPG biopsies and post-mortem tissue have recently identified mutations in PI3KCA, PTEN, TP53, ATM/MPL, histones and PDGF receptor overexpression. PI3KCA and PTEN mutations as well as PDGF receptor overexpression indicate upregulation of the PI3K/AKT/mTOR signaling axis, representing druggable targets. We recently developed a multifunctional kinase inhibitor (ST-182), which targets the PI3K/AKT/mTOR and MAPK pathways which is often upregulated in various malignancies as a compensatory mechanism when PI3K is inhibited. We evaluated ST-182's efficacy for targeting these pathways in patient derived DIPG by western blotting and reverse phase protein array analysis (RPPA). Phosphorylation changes of ERK and AKT, downstream signaling inhibition as well as diminished proliferation was shown in two DIPG cell lines (SU-DIPGIV and XIII) when treated with ST-182, indicating efficacy of co-targeting these pathways as a new therapeutic advance for DIPG. FACS analysis of DIPG cells (SU-DIPGXIII) identified a large percentage (>10%) of DIPG cells as ALDH positive indicating aggressive stem like features. Characterization of these distinct DIPG populations (ALDH+,-) at the transcriptome level was performed to understand differences in pathway signaling and to identify potential drug resistance mechanisms to ST-182. Utilizing an innovative transcriptome analysis approach, we identified elevated levels of MYC, E2F and DNA repair genes in ALDH+ cells, supporting stem like phenotype of ALDH+ DIPG cells. MYC has long been identified as a crucial player in maintaining embryonic stem cell pluripotency and self-renewal, whereas E2F provide transcriptional control of stem cell fate and DNA repair mechanisms maintain and regulate cancer stem cells. Pharmacological targeting of MAPK/PI3K/mTOR by ST-182 demonstrated up regulation of NFkB, apoptosis, hypoxia, p53 and inflammatory response in ALDH+ and ALDH- cells and down-regulation of MYC, E2F and DNA replication indicating efficacy of targeting these pathways in preventing/reversing stem-like phenotypes in the ALDH+ cell population. Our findings indicate efficacy of ST-182 for the treatment of ALDH+ cancer stem cells providing impetus for evaluation of molecularly targeted MAPK/PI3K/mTOR therapy. Our comprehensive transcriptome studies provide a new direction for the treatment of DIPG through novel insights into the underlying transcriptomic basis of drug resistant cancer stem cells. Development of new compounds such as ST-182 provides opportunities to implement precision medicine to improve treatment outcomes for DIPG patients. Citation Format: Stefanie Galbán, Carlos Espinoza, Karan Bedi, Uday B. Maachani, Mark M. Souweidane, Mats Ljungman, Marcian Van Dort, Brian D. Ross. Transcriptome profiles of cancer stem-like cells in patient-derived diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2073.

Pulling and Pushing Stem Cells to Control Their Differentiation.

Much has already been done to achieve precisely controlled and customised regenerative therapies. Thanks to recent advances made in several areas relevant to regenerative medicine including the use of stimuli-responsive materials, 4-dimensional biofabrication, inducible pluripotent stem cells, control of stem cell fate using chemical and physical factors, minimal access delivery, and information-communication technology. In this short perspective, recent advances are discussed with a focus on a recent report on the use of mechanical stretching of nanoparticle-laden stem cells by using external magnetic field to induce defined cardiac line differentiation. Although more and more tools are becoming available for engineering tissue models tissues and the range of potential applications is expanding, there is still much work to be done before it is proved to work with human cells, form tissues and ultimately achieve application in the clinic.

Orchestrating Ribosomal Subunit Coordination to Control Stem Cell Fate

The mechanisms responsible for maintaining ribosomal component stoichiometry, which is critical during cell fate transitions, are currently not well understood. In this issue of Cell Stem Cell, Corsini etal. (2018) demonstrate that the transcription and splicing-associated factor HTATSF1 controls stem cell fate by coordinately regulating ribosomal protein and RNA production.

MicroRNA-499a-5p Promotes Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells to Cardiomyocytes.

Since the adult mammalian heart has limited regenerative capacity, cardiac trauma, disease, and aging cause permanent loss of contractile tissue. This has fueled the development of stem cell-based strategies to provide the damaged heart with new cardiomyocytes. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are capable of self-renewal and differentiation into cardiomyocytes, albeit inefficiently. MicroRNAs (miRNAs, miRs) are non-coding RNAs that have the potential to control stem cell fate decisions and are employed in cardiac regeneration and repair. In this study, we tested the hypothesis that overexpression of miR-499a induces cardiomyogenic differentiation in BM-MSCs. Human BM-MSCs (hBM-MSCs) were transduced with lentiviral vectors encoding miR-499a-3p or miR-499a-5p and analyzed by immunostaining and western blotting methods 14days post-transduction. MiR-499a-5p-transduced cells adopted a polygonal/rod-shaped (myocyte-like) phenotype and showed an increase in the expression of the cardiomyocyte markers α-actinin and cTnI, as cardiogenic differentiation markers. These results indicate that miR-499a-5p overexpression promotes the cardiomyogenic differentiation of hBM-MSCs and may thereby increase their therapeutic efficiency in cardiac regeneration.

Microfluidic Encapsulation Supports Stem Cell Viability, Proliferation, and Neuronal Differentiation.

Stem cell encapsulation technology demonstrates much promise for the replacement of damaged tissue in several diseases, including spinal cord injury (SCI). The use of biocompatible microcapsules permits the control of stem cell fate in situ to facilitate the replacement of damaged/lost tissue. In this work, a novel customized microfluidic device was developed for the reproducible encapsulation of neural stem cells (NSCs) and dental pulp stem cells (DPSCs) within monodisperse, alginate-collagen microcapsules. Both cell types survived within the microcapsules for up to 21 days in culture. Stem cells demonstrated retention of their multipotency and neuronal differentiation properties upon selective release from the microcapsules, as demonstrated by high proliferation rates and the production of stem cell and neuronal lineage markers. When cell-laden microcapsules were transplanted into an organotypic SCI model, the microcapsules effectively retained the transplanted stem cells at the site of implantation. Implanted cells survived over a 10 day period in culture after transplantation and demonstrated commitment to a neural lineage. Our device provides a quick, effective, and aseptic method for the encapsulation of two different stem cell types (DPSCs and NSCs) within alginate-collagen microcapsules. Since stem cells were able to retain their viability and neural differentiation capacity within such microcapsules, this method provides a useful technique to study stem cell behavior within three-dimensional environments.

Dual non-viral gene delivery from microparticles within 3D high-density stem cell constructs for enhanced bone tissue engineering

High-density mesenchymal stem cell (MSC) aggregates can be guided to form bone-like tissue via endochondral ossification in vitro when culture media is supplemented with proteins, such as growth factors (GFs), to first guide the formation of a cartilage template, followed by culture with hypertrophic factors. Recent reports have recapitulated these results through the controlled spatiotemporal delivery of chondrogenic transforming growth factor-β1 (TGF-β1) and chondrogenic and osteogenic bone morphogenetic protein-2 (BMP-2) from microparticles embedded within human MSC aggregates to avoid diffusion limitations and the lengthy, costly in vitro culture necessary with repeat exogenous supplementation. However, since GFs have limited stability, localized gene delivery is a promising alternative to the use of proteins. Here, mineral-coated hydroxyapatite microparticles (MCM) capable of localized delivery of Lipofectamine-plasmid DNA (pDNA) nanocomplexes encoding for TGF-β1 (pTGF-β1) and BMP-2 (pBMP-2) were incorporated, alone or in combination, within MSC aggregates from three healthy porcine donors to induce sustained production of these transgenes. Three donor populations were investigated in this work due to the noted MSC donor-to-donor variability in differentiation capacity documented in the literature. Delivery of pBMP-2 within Donor 1 aggregates promoted chondrogenesis at week 2, followed by an enhanced osteogenic phenotype at week 4. Donor 2 and 3 aggregates did not promote robust glycosaminoglycan (GAG) production at week 2, but by week 4, Donor 2 aggregates with pTGF-β1/pBMP-2 and Donor 3 aggregates with both unloaded MCM and pBMP-2 enhanced osteogenesis compared to controls. These results demonstrate the ability to promote osteogenesis in stem cell aggregates through controlled, non-viral gene delivery within the cell masses. These findings also indicate the need to screen donor MSC regenerative potential in response to gene transfer prior to clinical application. Taken together, this work demonstrates a promising gene therapy approach to control stem cell fate in biomimetic 3D condensations for treatment of bone defects.

Physical Signals May Affect Mesenchymal Stem Cell Differentiation via Epigenetic Controls.

Marrow mesenchymal stem cells supply bone osteoblasts and adipocytes. Exercise effects to increase bone and decrease fat involve transfer of signals from the cytoplasm into the nucleus to regulate gene expression. We propose that exercise control of stem cell fate relies on structural connections that terminate in the nucleus and involve intranuclear actin structures that regulate epigenetic gene expression.

Bioengineering of the Human Neural Stem Cell Niche: A Regulatory Environment for Cell Fate and Potential Target for Neurotoxicity.

Human neural stem/progenitor cells of the developing and adult organisms are surrounded by the microenvironment, so-called neurogenic niche. The developmental processes of stem cells, such as survival, proliferation, differentiation, and fate decisions, are controlled by the mutual interactions between cells and the niche components. Such interactions are tissue specific and determined by the biochemical and biophysical properties of the niche constituencies and the presence of other cell types. This dynamic approach of the stem cell niche, when translated into in vitro settings, requires building up "biomimetic" microenvironments resembling natural conditions, where the stem/progenitor cell is provided with diverse extracellular signals exerted by soluble and structural cues, mimicking those found in vivo. The neural stem cell niche is characterized by a unique composition of soluble components including neurotransmitters and trophic factors as well as insoluble extracellular matrix proteins and proteoglycans. Biotechnological innovations provide tools such as a new generation of tunable biomaterials capable of releasing specific signals in a spatially and temporally controlled manner, thus creating in vitro nature-like conditions and, when combined with stem cell-derived tissue specific progenitors, producing differentiated neuronal tissue structures. In addition, substantial progress has been made on the protocols to obtain stem cell-derived cell aggregates such as neurospheres and self-assembled organoids.In this chapter, we have assessed the application of bioengineered human neural stem cell microenvironments to produce in vitro models of different levels of biological complexity for the efficient control of stem cell fate. Examples of biomaterial-supported two-dimensional and three-dimensional (2D and 3D) complex culture systems that provide artificial neural stem cell niches are discussed in the context of their application for basic research and neurotoxicity testing.

Small Molecule Antagonist of Cell Surface Glycosaminoglycans Restricts Mouse Embryonic Stem Cells in a Pluripotent State

Recently, the field of stem cell-based regeneration has turned its attention toward chemical approaches for controlling the pluripotency and differentiation of embryonic stem cells (ESCs) using drug-like small molecule modulators. Growth factor receptors or their associated downstream kinases that regulate intracellular signaling pathways during differentiation are typically the targets for these molecules. The glycocalyx, which plays an essential role in actuating responses to growth factors at the cellular boundary, offers an underexplored opportunity for intervention using small molecules to influence differentiation. Here, we show that surfen, an antagonist of cell-surface glycosaminoglycans required for growth factor association with cognate receptors, acts as a potent and general inhibitor of differentiation and promoter of pluripotency in mouse ESCs. This finding shows that drugging the stem cell Glycome with small molecules to silence differentiation cues can provide a powerful new alternative to existing techniques for controlling stem cell fate. Stem Cells 2018;36:45-54.

Directing Stem Cell Fate: The Synthetic Natural Product Connection.

Stem cells possess remarkable potential for the treatment of a broad array of diseases including many that lack therapeutic options. However, the use of cell-based products derived from stem cells as therapeutics has limitations including rejection, sufficient availability, and lack of appropriate engraftment. Chemical control of stem cells provides potential solutions for overcoming many of the current limitations in cell-based therapeutics. The development of exogenous molecules to control stem cell self-renewal or differentiation has arrived at natural product-based agents as an important class of modulators. The ex vivo production of cryopreserved cellular products for use in tissue repair is a relatively new area of medicine in which the conventional hurdles to implementing chemicals to effect human health are changed. Translational challenges centered on chemistry, such as pharmacokinetics, are reduced. Importantly, in many cases the desired human tissues can be evaluated against new chemicals, and approaches to cellular regulation can be validated in the clinically applicable system. As a result linking new and existing laboratory syntheses of natural products with findings of the compounds' unique abilities to regulate stem cell fate provides opportunities for developing improved methods for tissue manufacture, accessing probe compounds, and generating new leads that yield manufactured cells with improved properties. This review provides a summary of natural products that have shown promise in controlling stem cell fate and which have also been fully synthesized thereby providing chemistry platforms for further development.

Classification of Hydrogels Based on Their Source: A Review and Application in Stem Cell Regulation

Stem cells are recognized by their self-renewal ability and can give rise to specialized progeny. Hydrogels are an established class of biomaterials with the ability to control stem cell fate via mechanotransduction. They can mimic various physiological conditions to influence the fate of stem cells and are an ideal platform to support stem cell regulation. This review article provides a summary of recent advances in the application of different classes of hydrogels based on their source (e.g., natural, synthetic, or hybrid). This classification is important because the chemistry of substrate affects stem cell differentiation and proliferation. Natural and synthetic hydrogels have been widely used in stem cell regulation. Nevertheless, they have limitations that necessitate a new class of material. Hybrid hydrogels obtained by manipulation of the natural and synthetic ones can potentially overcome these limitations and shape the future of research in application of hydrogels in stem cell regulation.

AMPKα1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis.

Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPKα1-/- MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPKα1-/- MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1-/- phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1-/- MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.

Versatility of DGCR8 controls stem cell fate

MicroRNAs (miRNA) are a class of small non-coding RNAs, which control the expression of protein coding genes via translational repression and/or mRNA degradation. They regulate all biologic process...

Introduction of N-cadherin-binding motif to alginate hydrogels for controlled stem cell differentiation

Control of stem cell fate and phenotype using biomimetic synthetic extracellular matrices (ECMs) is an important tissue engineering approach. Many studies have focused on improving cell-matrix interactions. However, proper control of cell-cell interactions using synthetic ECMs could be critical for tissue engineering, especially with undifferentiated stem cells. In this study, alginate hydrogels were modified with a peptide derived from the low-density lipoprotein receptor-related protein 5 (LRP5), which is known to bind to N-cadherin, as a cell-cell interaction motif. In vitro changes in the morphology and differentiation of mouse bone marrow stromal cells (D1 stem cells) cultured in LRP5-alginate hydrogels were investigated. LRP5-alginate gels successfully induced stem cell aggregation and enhanced chondrogenic differentiation of D1 stem cells, compared to RGD-alginate gels, at low cell density. This approach to tailoring synthetic biomimetic ECMs using cell-cell interaction motifs may be critical in tissue engineering approaches using stem cells.

The metabolic programming of stem cells.

Advances in metabolomics have deepened our understanding of the roles that specific modes of metabolism play in programming stem cell fates. Here, we review recent metabolomic studies of stem cell metabolism that have revealed how metabolic pathways can convey changes in the extrinsic environment or their niche to program stem cell fates. The metabolic programming of stem cells represents a fine balance between the intrinsic needs of a cellular state and the constraints imposed by extrinsic conditions. A more complete understanding of these needs and constraints will afford us greater mastery over our control of stem cell fates.

Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells.

Mitochondrial dysfunction and endoplasmic reticulum stress (ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide (NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstrated the need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies.

NanoScript: A Versatile Nanoparticle-Based Synthetic Transcription Factor for Innovative Gene Manipulation.

Cellular reprogramming and stem cell-based therapies have shown tremendous potential in the field of regenerative medicine. To that end, developing tools to control stem cell fate is an attractive area of research for replacing damaged and diseased cells and reestablishing functional connections for tissue repair. Transcription factor (TFs) proteins are well known to regulate gene expression and direct stem cell fate. Inspired by natural TFs, NanoScript, a nanoparticle (NP)-based platform, mimics TFs to afford control over gene expression and stem cell fate for regenerative medicine. Here, we describe the construction of the NanoScript platform, which is designed with tunable properties to replicate the structure and function of TFs to bind to specific portions of the genome and regulate gene expression in a way that does not involve viral delivery.

Bioinspired Design of Polycaprolactone Composite Nanofibers as Artificial Bone Extracellular Matrix for Bone Regeneration Application.

The design and development of functional biomimetic systems for programmed stem cell response is a field of topical interest. To mimic bone extracellular matrix, we present an innovative strategy for constructing drug-loaded composite nanofibrous scaffolds in this study, which could integrate multiple cues from calcium phosphate mineral, bioactive molecule, and highly ordered fiber topography for the control of stem cell fate. Briefly, inspired by mussel adhesion mechanism, a polydopamine (pDA)-templated nanohydroxyapatite (tHA) was synthesized and then surface-functionalized with bone morphogenetic protein-7-derived peptides via catechol chemistry. Afterward, the resulting peptide-loaded tHA (tHA/pep) particles were blended with polycaprolactone (PCL) solution to fabricate electrospun hybrid nanofibers with random and aligned orientation. Our research demonstrated that the bioactivity of grafted peptides was retained in composite nanofibers. Compared to controls, PCL-tHA/pep composite nanofibers showed improved cytocompatibility. Moreover, the incorporated tHA/pep particles in nanofibers could further facilitate osteogenic differentiation potential of human mesenchymal stem cells (hMSCs). More importantly, the aligned PCL-tHA/pep composite nanofibers showed more osteogenic activity than did randomly oriented counterparts, even under nonosteoinductive conditions, indicating excellent performance of biomimetic design in cell fate decision. After in vivo implantation, the PCL-tHA/pep composite nanofibers with highly ordered structure could significantly promote the regeneration of lamellar-like bones in a rat calvarial critical-sized defect. Accordingly, the presented strategy in our work could be applied for a wide range of potential applications in not only bone regeneration application but also pharmaceutical science.

MiR-382-5p Controls Hematopoietic Stem Cell Differentiation Through the Downregulation of MXD1.

microRNAs are key regulators of gene expression that control stem cell fate by posttranscriptional downregulation of hundreds of target genes through seed pairing in their 3' untranslated region. In fact, miRNAs tightly regulate fundamental stem cell processes, like self-renewal, proliferation, and differentiation; therefore, miRNA deregulation may contribute to the development of solid tumors and hematological malignancies. miR-382-5p has been found to be upregulated in patients with myeloid neoplasms, but its role in normal hematopoiesis is still unknown. In this study, we demonstrated that miR-382-5p overexpression in CD34(+) hematopoietic stem/progenitor cells (HSPCs) leads to a significant decrease of megakaryocyte precursors coupled to increase of granulocyte ones. Furthermore, by means of a computational analysis using different prediction algorithms, we identified several putative mRNA targets of miR-382-5p that are downregulated upon miRNA overexpression (ie, FLI1, GATA2, MAF, MXD1, RUNX1, and SGK1). Among these, we validated MXD1 as real target of miR-382-5p by luciferase reporter assay. Finally, we showed that MXD1 knockdown mimics the effects of miR-382-5p overexpression on granulocyte and megakaryocyte differentiation of CD34(+) cells. Overall, our results demonstrated that miR-382-5p expression favors the expansion of granulocyte lineage and impairs megakaryocyte commitment through MXD1 downregulation. Therefore, our data showed for the first time that the miR-382-5p/MXD1 axis plays a critical role in myelopoiesis by affecting the lineage choice of CD34(+) HSPCs.