Stem Cell Lineage Specification Research Articles

Regenerative Engineering: Studies of the Rotator Cuff and other Musculoskeletal Soft Tissues

ABSTRACT‘Regenerative Engineering’ is the integration of advanced materials science, stem cell science, physics, developmental biology and clinical translation to regenerate complex tissues and organ systems. Advanced biomaterial and stem cell science converge as mechanisms to guide regeneration and the development of prescribed cell lineages from undifferentiated stem cell populations. Studies in somite development and tissue specification have provided significant insight into pathways of biological regulation responsible for tissue determination, especially morphogen gradients, and paracrine and contact-dependent signaling. The understanding of developmental biology mechanisms are shifting the biomaterial design paradigm by the incorporation of molecules into scaffold design and biomaterial development that are specifically targeted to promote the regeneration of soft tissues. Our understanding allows the selective control of cell sensitivity, and a temporal and spatial arrangement to modulate the wound healing mechanism, and the development of cell phenotype leading to the patterning of distinct and multi-scale tissue systems.Building on the development of mechanically compliant novel biomaterials, the integration of spatiotemporal control of biological, chemical and mechanical cues helps to modulate the stem cell niche and direct the differentiation of stem cell lineages. We have developed advanced biomaterials and biomimetic scaffold designs that can recapitulate the native tissue structure and mechanical compliance of soft musculoskeletal tissues, such as woven scaffold systems for ACL regeneration, non-woven scaffolds for rotator cuff tendon augmentation, and porous elastomers for regeneration of muscle tissue. Studies have clearly demonstrated the modulation of stem cell response to bulk biomaterial properties, such as toughness and elasticity, and scaffold structure, such as nanoscale and microscale dimensions. The integration of biological cues inspired from our understanding of developmental biology, along with chemical, mechanical and electrical stimulation drives our development of novel biomaterials aimed at specifying the stem cell lineage within 3-dimensional (3D) tissue systems. This talk will cover the development of biological cues, advanced biomaterials, and scaffold designs for the regeneration of complex soft musculoskeletal tissue systems such as ligament, tendon, and muscle.

Concise Review: Primary Cilia: Control Centers for Stem Cell Lineage Specification and Potential Targets for Cell-Based Therapies.

Directing stem cell lineage commitment prevails as the holy grail of translational stem cell research, particularly to those interested in the application of mesenchymal stem cells and adipose-derived stem cells in tissue engineering. However, elucidating the mechanisms underlying their phenotypic specification persists as an active area of research. In recent studies, the primary cilium structure has been intimately associated with defining cell phenotype, maintaining stemness, as well as functioning in a chemo, electro, and mechanosensory capacity in progenitor and committed cell types. Many hypothesize that the primary cilium may indeed be another important player in defining and controlling cell phenotype, concomitant with lineage-dictated cytoskeletal dynamics. Many of the studies on the primary cilium have emerged from disparate areas of biological research, and crosstalk amongst these areas of research is just beginning. To date, there has not been a thorough review of how primary cilia fit into the current paradigm of stem cell differentiation and this review aims to summarize the current cilia work in this context. The goal of this review is to highlight the cilium's function and integrate this knowledge into the working knowledge of stem cell biologists and tissue engineers developing regenerative medicine technologies. Stem Cells 2016;34:1445-1454.

The Lineage Specification of Mesenchymal Stem Cells Is Directed by the Rate of Fluid Shear Stress.

The effective regulation of fluid shear stress (FSS) on the lineage specification of mesenchymal stem cells (MSCs) remains to be addressed. We hypothesized that when MSCs are recruited to musculoskeletal system following stimulation, their differentiation into osteogenic or chondrogenic cells is directed by the rate of FSS (ΔSS) through modulation of the mechanosensitive, cation-selective channels (MSCCs), intracellular calcium levels, and F-actin. To this end, MSCs were exposed to laminar FSS linearly increased from 0 to 10 dyn/cm(2) in 0, 2, or 20 min and maintained at 10 dyn/cm(2) for a total of 20 min (termed as ΔSS 0-0', 0-2', and 0-20', respectively, representing more physiological (0-0') and non-physiological (0-2' and 0-20') ΔSS treatments). Our results showed 0-0' facilitated MSC differentiation towards chondrogenic and not osteogenic phenotype, by promoting moderate intracellular calcium concentration ([Ca(2+) ]i ) increase from the calcium channels with the exception of MSCCs or intracellular calcium stores, and F-actin organization. In contrast, 0-2' promoted MSCs towards osteogenic and not chondrogenic phenotype, by inducing significant [Ca(2+) ]i increase mainly from the MSCCs, and F-actin assembly. However, 0-20' elicited the modest osteogenic and chondrogenic phenotypes, as it induced the lowest [Ca(2+) ]i increase mainly from MSCCs, and F-actin assembly. Our results suggest that compared to the more physiological ΔSS, the non-physiological ΔSS favors [Ca(2+) ]i influx from MSCCs. An appropriate non-physiological ΔSS (0-2') even elicits a large [Ca(2+) ]i influx from the MSCCs that reverses the lineage specification of MSCs, providing validation for the high mechanosensitivity of MSCs and guidance for training osteoporosis and osteoarthritis patients. J. Cell. Physiol. 231: 1752-1760, 2016. © 2015 Wiley Periodicals, Inc.

Matrix elasticity-dependent differentiation of annulus fibrous-derived stem cells

Matrix elasticity-dependent differentiation of annulus fibrous-derived stem cells

Differentiation in Stem Cell Lineages and in Life: Explorations in the Male Germ Line Stem Cell Lineage.

I have been privileged to work on cellular differentiation during a great surge of discovery that has revealed the molecular mechanisms and genetic regulatory circuitry that control embryonic development and adult tissue maintenance and repair. Studying the regulation of proliferation and differentiation in the male germ line stem cell lineage has allowed us investigate how the developmental program imposes layers of additional controls on fundamental cellular processes like cell cycle progression and gene expression to give rise to the huge variety of specialized cell types in our bodies. We are beginning to understand how local signals from somatic support cells specify self-renewal versus differentiation in the stem cell niche at the apical tip of the testis. We are discovering the molecular events that block cell proliferation and initiate terminal differentiation at the switch from mitosis to meiosis-a signature event of the germ cell program. Our work is beginning to reveal how the developmental program that sets up the dramatic new cell type-specific transcription program that prepares germ cells for meiotic division and spermatid differentiation is turned on when cells become spermatocytes. I have had the privilege of working with incredible students, postdocs, and colleagues who have discovered, brainstormed, challenged, and refined our science and our ideas of how developmental pathways and cellular mechanisms work together to drive differentiation.

Directing lineage specification of human mesenchymal stem cells by decoupling electrical stimulation and physical patterning on unmodified graphene.

The organization and composition of the extracellular matrix (ECM) have been shown to impact the propagation of electrical signals in multiple tissue types. To date, many studies with electroactive biomaterial substrates have relied upon passive electrical stimulation of the ionic media to affect cell behavior. However, development of cell culture systems in which stimulation can be directly applied to the material - thereby isolating the signal to the cell-material interface and cell-cell contracts - would provide a more physiologically-relevant paradigm for investigating how electrical cues modulate lineage-specific stem cell differentiation. In the present study, we have employed unmodified, directly-stimulated, (un)patterned graphene as a cell culture substrate to investigate how extrinsic electrical cycling influences the differentiation of naïve human mesenchymal stem cells (hMSCs) without the bias of exogenous biochemicals. We first demonstrated that cyclic stimulation does not deteriorate the cell culture media or result in cytotoxic pH, which are critical experiments for correct interpretation of changes in cell behavior. We then measured how the expression of osteogenic and neurogenic lineage-specific markers were altered simply by exposure to electrical stimulation and/or physical patterns. Expression of the early osteogenic transcription factor RUNX2 was increased by electrical stimulation on all graphene substrates, but the mature marker osteopontin was only modulated when stimulation was combined with physical patterns. In contrast, the expression of the neurogenic markers MAP2 and β3-tubulin were enhanced in all electrical stimulation conditions, and were less responsive to the presence of patterns. These data indicate that specific combinations of non-biological inputs - material type, electrical stimulation, physical patterns - can regulate hMSC lineage specification. This study represents a substantial step in understanding how the interplay of electrophysical stimuli regulate stem cell behavior and helps to clarify the potential for graphene substrates in tissue engineering applications.

Cells Sensing Mechanical Cues: Stiffness Influences the Lifetime of Cell-Extracellular Matrix Interactions by Affecting the Loading Rate.

The question of how cells sense substrate mechanical cues has gained increasing attention among biologists. By introducing contour-based data analysis to single-cell force spectroscopy, we identified a loading-rate threshold for the integrin α2β1-DGEA bond beyond which a dramatic increase in bond lifetime was observed. On the basis of mechanical cues (elasticity or topography), the effective spring constant of substrates k is mapped to the loading rate r under actomyosin pulling speed v, which, in turn, affects the lifetime of the integrin-ligand bond. Additionally, downregulating v with a low-dose blebbistatin treatment promotes the neuronal lineage specification of mesenchymal stem cells on osteogenic stiff substrates. Thus, sensing of the loading rate is central to how cells sense mechanical cues that affect cell-extracellular matrix interactions and stem cell differentiation.

Epigenetic Modifying Drugs Inhibit MDS/AML Cell Growth through Selective Disruption of the Interactions Between Lineage-Determining Transcription Factors and DNA/Histone Modifiers

Normal hematopoiesis is controlled by a well-connected genetic network composed of several transcription factors (TFs) including PU.1 and GATA1. It has been postulated that both transcription factors and epigenetic modifiers work collaboratively to regulate hematopoietic stem cell differentiation and lineage specification as well as leukemogenesis. However, it is unclear about how the interplay between genetic network and epigenetic regulatory modifiers regulates locus-specific chromatin modifications and gene expression in normal hematopoiesis and hematologic malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Drugs targeting epigenetic modifiers including DNA methyltransferases (DNMTs), histone methyltransferases (HMTs) and histone deacetylases (HDACs) have been shown to be effective in a small portion of patients with MDS/AML, but the mechanisms underlying the efficacy and selectivity of different epigenetic modifying drugs are unknown. In this study, we performed growth-inhibition experiments with several epigenetic modifying drugs in multiple AML cell lines and identified two distinct lineage/differentiation-associated growth-inhibition patterns. Monocytic leukemia cells, but not erythroid leukemia cells, were sensitive to H3K4 HMT inhibitors, whereas both erythroid and monocytic leukemia cells were hypersensitive to DNMT and H3K27 HMT inhibitors. Importantly, co-immunoprecipitation experiments demonstrated lineage-specific interactions between the lineage-determining TFs (PU.1/SPI1 and GATA1) and the DNA/histone modifiers (DNMT1, DNMT3A/3B, TET2 and EZH2). Specifically, SPI1/PU.1 interacts with DNMT1 and EZH2, while GATA1 interacts with TET2 and DNMT3A/3B in MDS-derived erythroid leukaemia. In monocytic leukemia, SPI1/PU.1 interacts with TET2. Epigenetic modifying drugs such as azacytidine and 3-deazaneplanocin efficiently disrupted the interactions between the lineage-determining TFs and the DNA/histone modifiers without changing the expression of these proteins. We developed a new method, crosslink-assisted DNA modification immunoprecipitation assay (CDMIA), to simultaneously measure 5-methylcytosine (5-mC) and hydroxymethylcytosine (5-hmC). CDMIAs revealed significant drug-responsive changes in 5-mC/5-hmC at the promoters of differentiation/lineage-controlling genes such as PU.1/SPI1, but not at the global 5-mC/5-hmC. Sequential-ChIP and chromatin conformation capture (3C) showed that PU.1/SPI1 recruited polymerase II (pol-II) and the DNA/histone modifying complexes to PU.1/SPI1 toform distinct chromatin structures in a lineage-specific manner. We have selected azacytidine-resistant clones and established azacytidine-resistant cell lines from the previously azacytine-sensitive erythroid and monocytic leukemia cells. Strikingly, azacytine at the same concentrations failed to disrupt the interactions between the lineage-determining transcription factors and the DNA/histone modifiers in these drug-resistant leukemia cells. Genome-wide sequencing revealed novel mutations in TET2, TET3, DNMT3L and PU.1/SP1 that were confirmed by Sanger sequencing. These mutations correlated with the altered interactions between PU.1/SPI1 and the DNA/histone modifying complexes and predicted the responses to epigenetic modifying drugs. Examination of clinical specimens from patients with MDS/AML confirmed the presence of distinct lineage/differentiation-specific chromatin structure with a high-level recruitment of DNA/histone modifiers. Our genome-wide epigenetic analysis demonstrates the statistically significant enrichment of the SPI1/PU.1, TP53 and MYB DNA-binding motifs in hyper-H3K27 trimethylated DNA sequences in erythroid-predominant MDS. These results demonstrate the presence of locus-specific, drug-sensitive chromatin structures in MDS/AML subtypes. Our data revealed a novel epigenetic modifying drug action model that involves selective disruption of the disease-specific interactions between the lineage-determining factors and DNA/histone modifiers. Such drug action models may provide new insights into the mechanisms underlying the efficacy and selectivity of epigenetic modifying drugs. DisclosuresLarson:Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Ariad: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy.

Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s). In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC), a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs), spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

Skeletal muscle development in vertebrate animals

This review covers the pre- and post-natal development of skeletal muscle of vertebrate animals with cellular and molecular levels. The formation of skeletal muscle initiates from paraxial mesoderm during embryogenesis of individuals which develops somites and subsequently forms dermomyotome derived myotome to give rise axial musculature. This process (myogenesis) includes stem and progenitor cell maintenance, lineage specification, and terminal differentiation to form myofibrils consequent muscle fibers which control muscle mass and its multiplication. The main factors of muscle growth are proliferation and differentiation of myogenic cells in prenatal stage and also the growth of satellite cells at postnatal stage. There is no net increase in the number of muscle fibers in vertebrate animals after hatch or birth except fish. The development of muscle is characterized by hyperplasia and hypertrophy in prenatal and postnatal stages of individuals, respectively, through Wnt signalling pathway including environment, nutrition, sex, feed, growth and myogenic regulatory factors. Therefore further studies could elucidate new growth related genes, markers and factors to enhance meat production and enrich knowledge on muscle growth.Asian J. Med. Biol. Res. June 2015, 1(2): 139-148

A differential network analysis approach for lineage specifier prediction in stem cell subpopulations.

Background:Stem cell differentiation is a complex biological process. Cellular heterogeneity, such as the co-existence of different cell subpopulations within a population, partly hampers our understanding of this process. The modern single-cell gene expression technologies, such as single-cell RT-PCR and RNA-seq, have enabled us to elucidate such heterogeneous cell subpopulations. However, the identification of a transcriptional regulatory network (TRN) for each cell subpopulation within a population and genes determining specific cell fates (lineage specifiers) remains a challenge due to the slower development of appropriate computational and experimental workflows. Here, we propose a computational differential network analysis approach for predicting lineage specifiers in binary-fate differentiation events.Methods:The proposed method is based on a model that considers each stem cell subpopulation being in a stable state maintained by its specific TRN stability core, and cell differentiation involves changes in these stability cores between parental and daughter cell subpopulations. The method first reconstructs topologically different cell-subpopulation specific TRNs from single-cell gene expression data, literature knowledge and transcription factor (TF)–DNA binding-site prediction. Then, it systematically predicts lineage specifiers by identifying genes in the TRN stability cores in both parental and daughter cell subpopulations.Results:Application of this method to different stem cell differentiation systems was able to predict known and putative novel lineage specifiers. These examples include the differentiation of inner cell mass into either primitive endoderm or epiblast, different progenitor cells in the hematopoietic system, and the lung alveolar bipotential progenitor into either alveolar type 1 or alveolar type 2.Conclusions:The method is generally applicable to any binary-fate differentiation system, for which single-cell gene expression data are available. Therefore, it should aid in understanding stem cell lineage specification, and in the development of experimental strategies for regenerative medicine.

Calcineurin-NFAT Signaling Controls Somatic Cell Reprogramming in a Stage-Dependent Manner.

Calcineurin-NFAT signaling is critical for early lineage specification of mouse embryonic stem cells and early embryos. However, its roles in somatic cell reprogramming remain unknown. Here, we report that calcineurin-NFAT signaling has a dynamic activity and plays diverse roles at different stages of reprogramming. At the early stage, calcineurin-NFAT signaling is transiently activated and its activation is required for successful reprogramming. However, at the late stage of reprogramming, activation of calcineurin-NFAT signaling becomes a barrier for reprogramming and its inactivation is critical for successful induction of pluripotency. Mechanistically, calcineurin-NFAT signaling contributes to the reprogramming through regulating multiple early events during reprogramming, including mesenchymal to epithelial transition (MET), cell adhesion and emergence of SSEA1(+) intermediate cells. Collectively, this study reveals for the first time the important roles of calcineurin-NFAT signaling during somatic cell reprogramming and provides new insights into the molecular regulation of reprogramming.

Wnt signaling-mediated redox regulation maintains the germ line stem cell differentiation niche.

Adult stem cells continuously undergo self-renewal and generate differentiated cells. In the Drosophila ovary, two separate niches control germ line stem cell (GSC) self-renewal and differentiation processes. Compared to the self-renewing niche, relatively little is known about the maintenance and function of the differentiation niche. In this study, we show that the cellular redox state regulated by Wnt signaling is critical for the maintenance and function of the differentiation niche to promote GSC progeny differentiation. Defective Wnt signaling causes the loss of the differentiation niche and the upregulated BMP signaling in differentiated GSC progeny, thereby disrupting germ cell differentiation. Mechanistically, Wnt signaling controls the expression of multiple glutathione-S-transferase family genes and the cellular redox state. Finally, Wnt2 and Wnt4 function redundantly to maintain active Wnt signaling in the differentiation niche. Therefore, this study has revealed a novel strategy for Wnt signaling in regulating the cellular redox state and maintaining the differentiation niche.

Niche appropriation by Drosophila intestinal stem celltumours.

Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. We investigated Drosophila intestinal stem cell (ISC) tumors generated by suppressing Notch (N) signaling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumor initiation and an autocrine EGFR ligand, Spitz, during early tumor growth. Upon achieving a critical mass these tumors displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumor growth without the need for secondary mutations in growth signaling pathways. The appropriation of niche signaling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis.

Dynamic changes in replication timing and gene expression during lineage specification of human pluripotent stem cells.

Duplication of the genome in mammalian cells occurs in a defined temporal order referred to as its replication-timing (RT) program. RT changes dynamically during development, regulated in units of 400–800 kb referred to as replication domains (RDs). Changes in RT are generally coordinated with transcriptional competence and changes in subnuclear position. We generated genome-wide RT profiles for 26 distinct human cell types, including embryonic stem cell (hESC)-derived, primary cells and established cell lines representing intermediate stages of endoderm, mesoderm, ectoderm, and neural crest (NC) development. We identified clusters of RDs that replicate at unique times in each stage (RT signatures) and confirmed global consolidation of the genome into larger synchronously replicating segments during differentiation. Surprisingly, transcriptome data revealed that the well-accepted correlation between early replication and transcriptional activity was restricted to RT-constitutive genes, whereas two-thirds of the genes that switched RT during differentiation were strongly expressed when late replicating in one or more cell types. Closer inspection revealed that transcription of this class of genes was frequently restricted to the lineage in which the RT switch occurred, but was induced prior to a late-to-early RT switch and/or down-regulated after an early-to-late RT switch. Analysis of transcriptional regulatory networks showed that this class of genes contains strong regulators of genes that were only expressed when early replicating. These results provide intriguing new insight into the complex relationship between transcription and RT regulation during human development.

Neural Hedgehog signaling maintains stem cell renewal in the sensory touch dome epithelium

The touch dome is a highly patterned mechanosensory structure in the epidermis composed of specialized keratinocytes in juxtaposition with innervated Merkel cells. The touch dome epithelium is maintained by tissue-specific stem cells, but the signals that regulate the touch dome are not known. We identify touch dome stem cells that are unique among epidermal cells in their activated Hedgehog signaling and ability to maintain the touch dome as a distinct lineage compartment. Skin denervation reveals that renewal of touch dome stem cells requires a perineural microenvironment, and deleting Sonic hedgehog (Shh) in neurons or Smoothened in the epidermis demonstrates that Shh is an essential niche factor that maintains touch dome stem cells. Up-regulation of Hedgehog signaling results in neoplastic expansion of touch dome keratinocytes but no Merkel cell neoplasia. These findings demonstrate that nerve-derived Shh is a critical regulator of lineage-specific stem cells that maintain specialized sensory compartments in the epidermis.

Fluorescent hydrogels for embryoid body formation and osteogenic differentiation of embryonic stem cells.

Substrate mechanics (e.g., stiffness and topography of the microenvironment) are likely critical for driving normal morphogenesis and tissue development. As such, substrate mechanics imposed by hydrogels have been exploited to guide the lineage differentiation of stem cells and to drive stemness. In this work, we chemically modified gelatin hydrogels through glyceraldehyde cross-linking to render them suitable for cell culture. The modified hydrogels proved to be ideal for embryonic stem cell osteogenesis, initially providing a soft nonadhesive surface for the formation of embryoid bodies. They subsequently degraded in culture to afford a harder surface during osteoblast differentiation. The gels synthesized are highly fluorescent, relatively easy to prepare, and can potentially aid in overcoming the challenge of imaging changes to the microenvironments of cells during three-dimensional cell culture. Exploiting these materials could lead to the development of tissue-engineered products of increased complexity and rational treatment strategies.

Union is strength: matrix elasticity and microenvironmental factors codetermine stem cell differentiation fate

Stem cells are an attractive cellular source for regenerative medicine and tissue engineering applications due to their multipotency. Although the elasticity of the extracellular matrix (ECM) has been shown to have crucial impacts in directing stem cell differentiation, it is not the only contributing factor. Many researchers have recently attempted to design microenvironments that mimic the stem cell niche with combinations of ECM elasticity and other cues, such as ECM physical properties, soluble biochemical factors and cell-cell interactions, thereby driving cells towards their preferred lineages. Here, we briefly discuss the effect of matrix elasticity on stem cell lineage specification and then summarize recent advances in the study of the combined effects of ECM elasticity and other cues on the differentiation of stem cells, focusing on two aspects: biophysical and biochemical factors. In the future, biomedical scientists will continue investigating the union strength of matrix elasticity and microenvironmental cues for manipulating stem cell fates.

MicroRNA Profiling as Tool for Developmental Neurotoxicity Testing (DNT).

microRNAs (miRNAs) are small non-coding RNA molecules functioning as post-transcriptional regulators of gene expression. miRNAs play a significant role in organism development, regulating developmental timing, cell differentiation, and specification. In the developing brain, miRNAs regulate neural stem cell differentiation, lineage specification, synaptogenesis, and brain morphogenesis. Temporal and spatial specificity of miRNA expression make them an attractive marker to study cellular responses to toxicant exposure. Neural differentiation of murine embryonic stem cells (mESCs) has been established as an alternative method to study developmental neurotoxicity (DNT) in vitro. This unit will describe a method for miRNA profiling (miRNomics) as a molecular end point to study developmental neurotoxicity. A protocol for neural differentiation of mESC will be described as a cellular model for DNT testing. The miRNomics protocol is versatile and can be used with other DNT cellular systems such as primary cultures, human embryonic stem cells (hESCs), or induced pluripotent stem cells (iPSCs).

Molecular weight and concentration of heparin in hyaluronic acid-based matrices modulates growth factor retention kinetics and stem cell fate

Growth factors are critical for regulating and inducing various stem cell functions. To study the effects of growth factor delivery kinetics and presentation on stem cell fate, we developed a series of heparin-containing hyaluronic acid (HyA)-based hydrogels with various degrees of growth factor affinity and retention. To characterize this system, we investigated the effect of heparin molecular weight, fractionation, and relative concentration on the loading efficiency and retention kinetics of TGFβ1 as a model growth factor. At equal concentrations, high MW heparin both loaded and retained the greatest amount of TGFβ1, and had the slowest release kinetics, primarily due to the higher affinity with TGFβ1 compared to low MW or unfractionated heparin. Subsequently, we tested the effect of TGFβ1, presented from various heparin-containing matrices, to differentiate a versatile population of Sca-1+/CD45− cardiac progenitor cells (CPCs) into endothelial cells and form vascular-like networks in vitro. High MW heparin HyA hydrogels stimulated more robust differentiation of CPCs into endothelial cells, which formed vascular-like networks within the hydrogel. This observation was attributed to the ability of high MW heparin HyA hydrogels to sequester endogenously synthesized angiogenic factors within the matrix. These results demonstrate the importance of molecular weight, fractionation, and concentration of heparin on presentation of heparin-binding growth factors and their effect on stem cell differentiation and lineage specification.