Control Stem Cell Fate Research Articles

Control of Stem Cell Fate and Function by Polymer Nanofibers

Control of Stem Cell Fate and Function by Polymer Nanofibers

Material‐Driven Fibronectin Assembly Promotes Maintenance of Mesenchymal Stem Cell Phenotypes

Mesenchymal stem cells (MSCs) are a research tool to investigate fundamental biology and are candidates for use in regenerative medicine. In this context, significant efforts have been devoted to develop technologies to control stem cell fate, including the use of soluble factors in media. However, material properties offer alternative approaches that avoid the use of soluble factors. Here, a material system capable of sustaining the growth of stem cells (maintaining stemness) and of promoting highly efficient differentiation upon external stimulation is described. Poly(ethyl acrylate) induces assembly of fibronectin (FN) into nanonetworks (FN fibrillogenesis) upon simple adsorption from solutions. It is shown that these FN nanonetworks allow growth of MSCs and maintenance of stemness for long periods of time (up to 30 d) using basal media in absence of soluble factors. Additionally, the system promotes enhanced levels of differentiation when defined supplemented media are used. The study reveals the critical role of the intermediate protein layer at the material interface to control MSC fate regardless of the properties of the underlying material and it introduces a new material system as a candidate to be used in MSC niche design.

Fractones: extracellular matrix niche controlling stem cell fate and growth factor activity in the brain in health and disease.

The stem cell niche refers to a specific microenvironment where stem cells proliferate and differentiate to produce new specialized cells throughout an organism's adulthood. Growth factors are crucial signaling molecules that diffuse through the extracellular space, reach the stem cell niche, and ultimately promote stem cell proliferation and differentiation. However, it is not well known how multiple growth factors, often with antagonistic activities, work together in the stem cell niche to select target stem cell populations and determine stem cell fate. There is accumulating evidence suggesting that extracellular matrix (ECM) molecules play an important role in promoting growth factor access and activity in the stem cell niche. In the adult brain neurogenic zone, where neural stem cells (NSCs) reside, there exist specialized ECM structures, which we have named fractones. The processes of NSC allow them to come into contact with fractones and interact with its individual components, which include heparan sulfate proteoglycans (HSPGs) and laminins. We have demonstrated that fractone-associated HSPGs bind growth factors and regulate NSC proliferation in the neurogenic zone. Moreover, emerging results show that fractones are structurally altered in animal models with autism and adult hydrocephalus, as demonstrated by changes in fractone size, quantity, or HSPG content. Interestingly, ECM structures similar to fractones have been found throughout β-amyloid plaques in the brain of patients with Alzheimer's disease. Pathological fractones may cause imbalances in growth factor activity and impair neurogenesis, leading to inflammation and disorder. Generally speaking, these stem cell niche structures play a potentially vital role in controlling growth factor activity during both health and disease.

RhoA inhibits neural differentiation in murine stem cells through multiple mechanisms.

Spontaneous neural differentiation of embryonic stem cells is induced by Noggin-mediated inhibition of bone morphogenetic protein 4 (BMP4) signaling. RhoA is a guanosine triphosphatase (GTPase) that regulates cytoskeletal dynamics and gene expression, both of which control stem cell fate. We found that disruption of Syx, a gene encoding a RhoA-specific guanine nucleotide exchange factor, accelerated retinoic acid-induced neural differentiation in murine embryonic stem cells aggregated into embryoid bodies. Cells from Syx(+/+) and Syx(-/-) embryoid bodies had different abundances of proteins implicated in stem cell pluripotency. The differentiation-promoting proteins Noggin and RARγ (a retinoic acid receptor) were more abundant in cells of Syx(-/-) embryoid bodies, whereas the differentiation-suppressing proteins SIRT1 (a protein deacetylase) and the phosphorylated form of SMAD1 (the active form of this transcription factor) were more abundant in cells of Syx(+/+) embryoid bodies. These differences were blocked by the overexpression of constitutively active RhoA, indicating that the abundance of these proteins was maintained, at least in part, by RhoA activity. The peripheral stress fibers in cells from Syx(-/-) embryoid bodies were thinner than those in Syx(+/+) cells. Furthermore, less Noggin and fewer vesicles containing Rab3d, a GTPase that mediates Noggin trafficking, were detected in cells from Syx(-/-) embryoid bodies, which could result from increased Noggin exocytosis. These results suggested that, in addition to inhibiting Noggin transcription, RhoA activity in wild-type murine embryonic stem cells also prevented neural differentiation by limiting Noggin secretion.

Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineagecommitment.

Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. We show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. Our results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis.

3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds.

Tissue-specific patterned stem cell differentiation serves as the basis for the development, remodeling, and regeneration of the multicellular structure of the native tissues. We herein proposed a cytocompatible 3D casting process to recapitulate this patterned stem cell differentiation for reconstructing multicellular tissues in vitro. We first reconstituted the 2D culture conditions for stem cell fate control within 3D hydrogel by incorporating the sets of the diffusible signal molecules delivered through drug-releasing microparticles. Then, utilizing thermo-responsivity of methylcellulose (MC), we developed a cytocompatible casting process to mold these hydrogels into specific 3D configurations, generating the targeted spatial gradients of diffusible signal molecules. The liquid phase of the MC solution was viscous enough to adopt the shapes of 3D impression patterns, while the gelated MC served as a reliable mold for patterning the hydrogel prepolymers. When these patterned hydrogels were integrated together, the stem cells in each hydrogel distinctly differentiated toward individually defined fates, resulting in the formation of the multicellular tissue structure bearing the very structural integrity and characteristics as seen in vascularized bones and osteochondral tissues.

Characterisation of minimalist co-assembled fluorenylmethyloxycarbonyl self-assembling peptide systems for presentation of multiple bioactive peptides

The nanofibrillar structures that underpin self-assembling peptide (SAP) hydrogels offer great potential for the development of finely tuned cellular microenvironments suitable for tissue engineering. However, biofunctionalisation without disruption of the assembly remains a key issue. SAPS present the peptide sequence within their structure, and studies to date have typically focused on including a single biological motif, resulting in chemically and biologically homogenous scaffolds. This limits the utility of these systems, as they cannot effectively mimic the complexity of the multicomponent extracellular matrix (ECM). In this work, we demonstrate the first successful co-assembly of two biologically active SAPs to form a coassembled scaffold of distinct two-component nanofibrils, and demonstrate that this approach is more bioactive than either of the individual systems alone. Here, we use two bioinspired SAPs from two key ECM proteins: Fmoc-FRGDF containing the RGD sequence from fibronectin and Fmoc-DIKVAV containing the IKVAV sequence from laminin. Our results demonstrate that these SAPs are able to co-assemble to form stable hybrid nanofibres containing dual epitopes. Comparison of the co-assembled SAP system to the individual SAP hydrogels and to a mixed system (composed of the two hydrogels mixed together post-assembly) demonstrates its superior stable, transparent, shear-thinning hydrogels at biological pH, ideal characteristics for tissue engineering applications. Importantly, we show that only the coassembled hydrogel is able to induce in vitro multinucleate myotube formation with C2C12 cells. This work illustrates the importance of tissue engineering scaffold functionalisation and the need to develop increasingly advanced multicomponent systems for effective ECM mimicry. Statement of SignificanceSuccessful control of stem cell fate in tissue engineering applications requires the use of sophisticated scaffolds that deliver biological signals to guide growth and differentiation. The complexity of such processes necessitates the presentation of multiple signals in order to effectively mimic the native extracellular matrix (ECM). Here, we establish the use of two biofunctional, minimalist self-assembling peptides (SAPs) to construct the first co-assembled SAP scaffold. Our work characterises this construct, demonstrating that the physical, chemical, and biological properties of the peptides are maintained during the co-assembly process. Importantly, the coassembled system demonstrates superior biological performance relative to the individual SAPs, highlighting the importance of complex ECM mimicry. This work has important implications for future tissue engineering studies.

CLE peptide signaling and nitrogen interactions in plant root development.

The CLAVATA signaling pathway is essential for the regulation of meristem activities in plants. This signaling pathway consists of small signaling peptides of the CLE family interacting with CLAVATA1 and leucine-rich repeat receptor-like kinases (LRR-RLKs). The peptide-receptor relationships determine the specificities of CLE-dependent signals controlling stem cell fate and differentiation that are critical for the establishment and maintenance of shoot and root apical meristems. Plants root systems are highly organized into three-dimensional structures for successful anchoring and uptake of water and mineral nutrients from the soil environment. Recent studies have provided evidence that CLE peptides and CLAVATA signaling pathways play pivotal roles in the regulation of lateral root development and systemic autoregulation of nodulation (AON) integrated with nitrogen (N) signaling mechanisms. Integrations of CLE and N signaling pathways through shoot-root vascular connections suggest that N demand modulates morphological control mechanisms and optimize N uptake as well as symbiotic N fixation in roots.

IGF-1 C Domain-Modified Hydrogel Enhances Cell Therapy for AKI.

Low cell retention and engraftment after transplantation limit the successful application of stem cell therapy for AKI. Engineered microenvironments consisting of a hydrogel matrix and growth factors have been increasingly successful in controlling stem cell fate by mimicking native stem cell niche components. Here, we synthesized a bioactive hydrogel by immobilizing the C domain peptide of IGF-1 (IGF-1C) on chitosan, and we hypothesized that this hydrogel could provide a favorable niche for adipose-derived mesenchymal stem cells (ADSCs) and thereby enhance cell survival in an AKI model. In vitro studies demonstrated that compared with no hydrogel or chitosan hydrogel only, the chitosan-IGF-1C hydrogel increased cell viability through paracrine effects. In vivo, cotransplantation of the chitosan-IGF-1C hydrogel and ADSCs in ischemic kidneys ameliorated renal function, likely by the observed promotion of stem cell survival and angiogenesis, as visualized by bioluminescence imaging and attenuation of fibrosis. In conclusion, IGF-1C immobilized on a chitosan hydrogel provides an artificial microenvironment for ADSCs and may be a promising therapeutic approach for AKI.

The CLAVATA signaling pathway mediating stem cell fate in shoot meristems requires Ca(2+) as a secondary cytosolic messenger.

CLAVATA1 (CLV1) is a receptor protein expressed in the shoot apical meristem (SAM) that translates perception of a non-cell-autonomous CLAVATA3 (CLV3) peptide signal into altered stem cell fate. CLV3 reduces expression of WUSCHEL (WUS) and FANTASTIC FOUR 2 (FAF2) in the SAM. Expression of WUS and FAF2 leads to maintenance of undifferentiated stem cells in the SAM. CLV3 binding to CLV1 inhibits expression of these genes and controls stem cell fate in the SAM through an unidentified signaling pathway. Cytosolic Ca(2+) elevations, cyclic nucleotide (cGMP)-activated Ca(2+) channels, and cGMP have been linked to signaling downstream of receptors similar to CLV1. Hence, we hypothesized that cytosolic Ca(2+) elevation mediates the CLV3 ligand/CLV1 receptor signaling that controls meristem stem cell fate. CLV3 application to Arabidopsis seedlings results in elevation of cytosolic Ca(2+) and cGMP. CLV3 control of WUS was prevented in a genotype lacking a functional cGMP-activated Ca(2+) channel. In wild-type plants, CLV3 inhibition of WUS and FAF2 expression was impaired by treatment with either a Ca(2+) channel blocker or a guanylyl cyclase inhibitor. When CLV3-dependent repression of WUS is blocked, altered control of stem cell fate leads to an increase in SAM size; we observed a larger SAM size in seedlings treated with the Ca(2+) channel blocker. These results suggest that the CLV3 ligand/CLV1 receptor system initiates a signaling cascade that elevates cytosolic Ca(2+), and that this cytosolic secondary messenger is involved in the signal transduction cascade linking CLV3/CLV1 to control of gene expression and stem cell fate in the SAM.

Abstract IA26: Long-term single cell quantification: New tools for old questions

Abstract Stem cell systems are highly complex and dynamic, and consist of large numbers of different cells expressing many molecules. Despite intensive research, many long-standing questions in stem cell research remain disputed. One major reason is the fact that we usually only analyze populations of cells - rather than individual cells - at very few time points of an experiment. Tracking of individual cells would be an extremely powerful approach to improve our understanding of molecular cell fate control. We are therefore developing imaging systems to follow the fate of individual cells over many generations. We program new software to help recording and displaying the divisional history, position, properties, interaction, etc. of all individual cells over many generations. Our approaches also allow continuous long-term quantification of protein expression or activity in individual living cells. The resulting novel kind of continuous quantitative single cell data is used for the generation of improved models describing the molecular control of stem cell fates. I will discuss how we try to find answers for long standing questions in stem cell research. Citation Format: Timm Schroeder. Long-term single cell quantification: New tools for old questions. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA26.

Bio-inspired polymer surfaces for reverse transfection of siRNA to enhance osteogenic differentiation and bone formation of human adipose-derived stem cells

Event Abstract Back to Event Bio-inspired polymer surfaces for reverse transfection of siRNA to enhance osteogenic differentiation and bone formation of human adipose-derived stem cells Jisoo Shin1, Jung-Ho Cho1, Yoonhee Jin1, Hyun-Ji Park1, Jong Seung Lee1, Kisuk Yang1 and Seung-Woo Cho1 1 Yonsei University, Department of Biotechnology, Korea Introduction: Controlling stem cell fate by regulating gene expression using small interfering RNA (siRNA) has been recently studied. However, conventional siRNA transfection methods are not efficient to induce guided stem cell differentiation due to low intracellular transfer of siRNA and short target silencing. To overcome these limitations, we developed bio-inspired polymer surfaces for reverse transfection of siRNA. We efficiently immobilized siRNA onto the catechol-modified surface of biodegradable polymer scaffolds to transfect siRNA into human stem cells. We tested whether this system could offer enhanced, long-term siRNA silencing to stem cells for promoting osteogenic differentiation and bone formation of stem cells. Materials and Methods: To prepare bio-inspired scaffolds for siRNA reverse transfection, biodegradable poly(lactide-co-glycolide) (PLGA) film was coated with catechol group, known as a main adhesive component of mussel pads. Then siRNA complexed with lipidoid nanoparticles was immobilized onto the polymer surfaces for non-viral gene delivery. Human adipose-derived stem cells (hADSCs) were seeded onto this scaffold system to reverse transfect siRNA targeting an osteogenic differentiation inhibitor to enhance osteogenic differentiation of hADSCs. Finally, hADSCs differentiated onto the siRNA-immobilized scaffolds were transplanted into critical-sized calvarial bone defects for in vivo bone formation. Results and Discussion: Immobilization efficiency of siRNA-lipidoid complexes on the catechol-modified surfaces was more than 90%. Target knockdown efficacy in hADSCs cultured on the surfaces modified with siRNA-lipidoid was around 90% with no significant cytotoxicity. The expression of osteogenic markers (OPN and ColI) was significantly upregulated in hADSCs differentiated on the surfaces with siRNA targeting osteogenic differentiation suppressor in vitro. Finally, we confirmed transplantation of hADSCs with siRNA-lipidoid immobilized PLGA scaffolds significantly enhanced in vivo bone formation in critical-sized calvarial bone defects confirmed by micro-CT and immunohistochemistry analysis. Conclusion: In summary, we developed bio-inspired polymer surfaces for siRNA reverse transfection platform exhibiting highly effective intracellular siRNA transfer and long-lasting target silencing. The polymer surfaces developed in this study could provide efficient genetic manipulation for improving stem cell engineering and tissue regeneration. This work was supported by a grant (HI14C1588) from the Korea Health Technology R&D Project funded by the Ministry of Health and Welfare, Republic of Korea; This work was supported by the Brain Korea 21 plus (BK21PLUS) program. J. Shin is a fellowship awardee by BK21 plus program Keywords: Bone Regeneration, stem cell, Surface modification, siRNA delivery Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in constructing tissue substitutes Citation: Shin J, Cho J, Jin Y, Park H, Lee J, Yang K and Cho S (2016). Bio-inspired polymer surfaces for reverse transfection of siRNA to enhance osteogenic differentiation and bone formation of human adipose-derived stem cells. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00670 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jisoo Shin Jung-Ho Cho Yoonhee Jin Hyun-Ji Park Jong Seung Lee Kisuk Yang Seung-Woo Cho Google Jisoo Shin Jung-Ho Cho Yoonhee Jin Hyun-Ji Park Jong Seung Lee Kisuk Yang Seung-Woo Cho Google Scholar Jisoo Shin Jung-Ho Cho Yoonhee Jin Hyun-Ji Park Jong Seung Lee Kisuk Yang Seung-Woo Cho PubMed Jisoo Shin Jung-Ho Cho Yoonhee Jin Hyun-Ji Park Jong Seung Lee Kisuk Yang Seung-Woo Cho Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Cadherin-matrix engineering for homogeneous reaction control of ES/iPS/Stem Cells

Event Abstract Back to Event Cadherin-matrix engineering for homogeneous reaction control of ES/iPS/Stem Cells Toshihiro Akaike1 1 Foundation for Advancement of International Science, Biomaterials Center for Regenerative Medical Engineering, Japan Recently, embryonic stem (ES) and induced pluripotent stem (iPS) cells have shown remarkable potential to treat human diseases. Biomaterials are rapidly being developed as powerful artificial microenvironments to study and control stem-cell fate, such as proliferation and differentiation. Our recent advances include biomaterial-based artificial extracellular matrix formed by immobilizing cell-recognizable molecule, growth factors and cytokines. Here, we propose “Cadherin-Matrix Engineering” for new frontier of cell-recognizable biomaterials for construction of “Cell-cooking plate” for ES/iPS cells technology using chimeric proteins of cell adhesion molecules (e.g., E-cadherin, N-cadherin, or VE-cadherin). Our novel E-cadherin-based engineered extracellular matrix showed fascinating results: (1) highly homogeneous differentiation of definitive endoderm cells under single-cell level; (2) uniform distribution of growth factors resulted in con differentiation even in lower concentration of free culture due to the absence of serum and feeder layers; (3) completely defined and xeno- short period of differentiation; (4) highly functional hepatocytes within soluble factors; (5) striking effect of matrix-dependent cell sorting for isolation and enrichment of mature hepatocytes for possible elimination of contaminated and poorly differentiated cells; (6) the unique opportunity for continuous monitoring of cellular behavior in different stages of differentiation. Taken together, our novel recombinant ECM is advantageous for generating homogeneous population of differentiated cells without any enzymatic stress and cell sorting, suggesting that the improved method of differentiation is highly promising for clinically significant adult cells (hepatocytes, neural cells, cardiomyocytes, pancreatic cells and the likes ). We established a novel biomedical field in applied cadherin biology named as “Cadherin-Matrix Engineering” which can be applied to “Cell-cooking Plate” for ES/iPS and other stem cells in regenerative medicine. Keywords: Cell Differentiation, Extracellular Matrix, Regenerative Medicine, biomimetic culture Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials and cellular signaling Citation: Akaike T (2016). Cadherin-matrix engineering for homogeneous reaction control of ES/iPS/Stem Cells. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00223 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Toshihiro Akaike Google Toshihiro Akaike Google Scholar Toshihiro Akaike PubMed Toshihiro Akaike Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Harnessing isomerization-mediated manipulation of nonspecific cell/matrix interactions to reversibly trigger and suspend stem cell differentiation.

Specific protein-cell and drug-cell interactions have been widely used to manipulate stem cell fate. Despite extensive studies, most current platforms cannot realize reversible manipulation of stem cell differentiation. In this work, we report a photodynamic zwitterionic hydrogel capable of reversibly triggering and suspending the differentiation process via manipulating nonspecific interactions between cultured stem cells and the hydrogel. The differentiation state of stem cells can be altered by exposing the hydrogel to a selected light program, while differentiation can be immediately suspended when near-infrared exposure converts the hydrogel into a purely zwitterionic form. While many other studies apply specific interactions to control stem cell fate, this work provides a completely different approach-allowing reversible, real-time and localized manipulation of stem cell fate choice via nonspecific interactions.

Role of Polyunsaturated Fatty Acids and Their Metabolites on Stem Cell Proliferation and Differentiation.

The nervous system is highly enriched with long-chain polyunsaturated fatty acids (PUFAs). Essential fatty acids, namely, ω-6 (n - 6) and ω-3 (n - 3) PUFA, and their metabolites are critical components of cell structure and function and could therefore influence stem cell fate. The available supporting experimental data reveal that n - 6 and n - 3 PUFA and their metabolites can act through multiple mechanisms to promote the proliferation and differentiation of various stem cell types. PUFAs and their mediators regulate several processes within the brain, such as neurotransmission, cell survival and neuroinflammation, and thereby mood and cognition. PUFA levels and the signaling pathways that they regulate are altered in various neurological disorders, including Alzheimer's disease and major depression. Therefore, elucidating the role of PUFAs and their metabolites in stem cell fate regulation is important for stem cell biology as well as stem cell therapy. PUFA-based interventions to generate a positive environment for stem cell proliferation or differentiation might be a promising and practical approach to controlling stem cell fate for clinical applications.

Mechanisms Regulating Spermatogonial Differentiation.

Mammalian spermatogenesis is a complex and highly ordered process by which male germ cells proceed through a series of differentiation steps to produce haploid flagellated spermatozoa. Underlying this process is a pool of adult stem cells, the spermatogonial stem cells (SSCs), which commence the spermatogenic lineage by undertaking a differentiation fate decision to become progenitor spermatogonia. Subsequently, progenitors acquire a differentiating spermatogonia phenotype and undergo a series of amplifying mitoses while becoming competent to enter meiosis. After spermatocytes complete meiosis, post-meiotic spermatids must then undergo a remarkable transformation from small round spermatids to a flagellated spermatozoa with extremely compacted nuclei. This chapter reviews the current literature pertaining to spermatogonial differentiation with an emphasis on the mechanisms controlling stem cell fate decisions and early differentiation events in the life of a spermatogonium.

OS1413 光造形法により作製したマイクロパターンによる細胞機能制御

Cells can adapt their biological behaviors due to changes in extracellular matrix topology. Previous studies have demonstrated the use of micropatterns (MPs) to control stem cell fate. However, such studies synthesized the MPs using photolithography techniques, which limited the structures to be simple and does not fully represent the native environment. In our study, we have developed a novel stereolithographical system using two-photon excitation, and fabricated microgrooves with circular, trapezoidal, triangular, or square contours. When preosteoblasts were cultured on the MPs, the cytoskeletons were remodeled differently resulting in varying cell morphologies depending on the contour. Localization of the cell nuclei was also observed to be influence by substrate topology, which may hint at connections to cell differentiation.

Fabrication of Elasticity-Tunable Gelatinous Gel for Mesenchymal Stem Cell Culture.

Surface elasticity or stiffness of an underlying substrate may regulate cellular functions such as adhesion, proliferation, signaling, differentiation, and migration. Recent studies have reported on the development of biomaterials to control stem cell fate determination via the stiffness of the culture substrates. In this chapter, we provide a detailed protocol for fabricating elasticity-tunable gelatinous hydrogels for stem cell culture with photo-induced or thermo-induced crosslinking of well-developed styrenated gelatin (StG). We also include the detailed application of gelatinous gel for mesenchymal stem cell (MSC) culture and sample collection for transcriptional and proteomic analysis.

Ordered self-assembled monolayers terminated with different chemical functional groups direct neural stem cell linage behaviours

Neural stem cells (NSCs) have been a promising candidate for stem cell-based nerve tissue regeneration. Therefore, the design of idea biomaterials that deliver precise regulatory signals to control stem cell fate is currently a crucial issue that depends on a profound understanding of the interactions between NSCs with the surrounding micro-environment. In this work, self-assembled monolayers of alkanethiols on gold with different chemical groups, including hydroxyl (−OH), amino (−NH2), carboxyl (−COOH) and methyl (−CH3), were used as a simple model to study the effects of surface chemistry on NSC fate decisions. Contact angle measurement and x-ray photoelectron spectroscopy (XPS) examination implied that all types of alkanethiols self-assembled on gold into a close-packed phase structure with similar molecular densities. In this study, we evaluated NSC adhesion, migration and differentiation in response to different chemical functional groups cultured under serum-free conditions. Our studies showed that NSCs exhibited certain phenotypes with extreme sensitivity to surface chemical groups. Compared with other functional groups, the SAMs with hydroxyl end-groups provided the best micro-environment in promoting NSC migration and maintaining an undifferentiated or neuronal differentiation state. −NH2 surfaces directed neural stem cells into astrocytic lineages, while NSCs on −COOH and −CH3 surfaces had a similar potency to differentiate into three nerve lineages. To further investigate the possible signaling pathway, the gene expression of integrin β1 and β4 were examined. The results indicated that a high expression of β1 integrin would probably have a tight correlation with the expression of nestin, which implied the stemness of NSCs, while β4 integrin seemed to correspond to the differentiated NSCs. The results presented here give useful information for the future design of biomaterials to regulate the preservation, proliferation and differentiation of NSCs for central nervous tissue engineering.

Stem Cell Ribonomics: RNA-Binding Proteins and Gene Networks in Stem Cell Differentiation.

Stem cells are undifferentiated cells with the ability to self-renew and the potential to differentiate into all body cell types. Stem cells follow a developmental genetic program and are able to respond to alterations in the environment through various signaling pathways. The mechanisms that control these processes involve the activation of transcription followed by a series of post-transcriptional events. These post-transcriptional steps are mediated by the interaction of RNA-binding proteins (RBPs) with defined subpopulations of RNAs creating a regulatory gene network. Characterizing these RNA-protein networks is essential to understanding the regulatory mechanisms underlying the control of stem cell fate. Ribonomics is the combination of classical biochemical purification protocols with the high-throughput identification of transcripts applied to the functional characterization of RNA-protein complexes. Here, we describe the different approaches that can be used in a ribonomic approach and how they have contributed to understanding the function of several RBPs with central roles in stem cell biology.