hPS Cell Research Articles

Generation and application of novel hES cell reporter lines for the differentiation and maturation of hPS cell-derived islet-like clusters

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional β-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of β-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as β-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal β-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.

STRAIGHT-IN: a platform for rapidly generating panels of genetically modified human pluripotent stem cell lines.

Targeted integration of large DNA cargoes (>10 kb) or genomic replacements in mammalian cells, such as human pluripotent stem cells (hPS cells), remains challenging. Here we describe a platform termed serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation (STRAIGHT-IN) to circumvent this. First, a landing pad cassette is precisely inserted or used to replace specific genomic regions. The site-specific integrase Bxb1 then enables DNA constructs, including those >50 kb, to be integrated into the genome, while Cre recombinase excises auxiliary DNA sequences to prevent postintegrative silencing. Using a strategy whereby the positive selection marker is only expressed if the donor plasmid carrying the payload is correctly targeted, we can obtain 100% enrichment for cells containing the DNA payload. Procedures for expressing Cre efficiently also mean that a clonal isolation step is no longer essential to derive the required genetically modified hPS cells containing the integrated DNA, potentially reducing clonal variability. Furthermore, STRAIGHT-IN facilitates rapid and multiplexed generation of genetically matched hPS cells when multiple donor plasmids are delivered simultaneously. STRAIGHT-IN has various applications, which include integrating complex genetic circuits for synthetic biology, as well as creating panels of hPS cells lines containing, as necessary, hundreds of disease-linked variants for disease modeling and drug discovery. After establishing the hPS cell line containing the landing pad, the entire procedure, including donor plasmid synthesis, takes 1.5-3 months, depending on whether single or multiple DNA payloads are integrated. This protocol only requires the researcher to be skilled in molecular biology and standard cell culture techniques.

Integrating population genetics, stem cell biology and cellular genomics to study complex human diseases.

Human pluripotent stem (hPS) cells can, in theory, be differentiated into any cell type, making them a powerful in vitro model for human biology. Recent technological advances have facilitated large-scale hPS cell studies that allow investigation of the genetic regulation of molecular phenotypes and their contribution to high-order phenotypes such as human disease. Integrating hPS cells with single-cell sequencing makes identifying context-dependent genetic effects during cell development or upon experimental manipulation possible. Here we discuss how the intersection of stem cell biology, population genetics and cellular genomics can help resolve the functional consequences of human genetic variation. We examine the critical challenges of integrating these fields and approaches to scaling them cost-effectively and practically. We highlight two areas of human biology that can particularly benefit from population-scale hPS cell studies, elucidating mechanisms underlying complex disease risk loci and evaluating relationships between common genetic variation and pharmacotherapeutic phenotypes.

Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions

The unlimited expansion of human progenitor cells in vitro could unlock many prospects for regenerative medicine. However, it remains an important challenge as it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from those mechanisms promoting their differentiation. This study focuses on the expansion of human pluripotent stem (hPS) cell-derived pancreatic progenitors (PP) to advance novel therapies for diabetes. We obtained mechanistic insights into PP expansion requirements and identified conditions for the robust and unlimited expansion of hPS cell-derived PP cells under GMP-compliant conditions through a hypothesis-driven iterative approach. We show that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling, and inhibition of selected branches of the TGFβ and Wnt signaling pathways are necessary for the effective decoupling of PP proliferation from differentiation. This enabled the reproducible, 2000-fold, over 10 passages and 40–45 d, expansion of PDX1+/SOX9+/NKX6-1+ PP cells. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. Using these conditions, PDX1+/SOX9+/NKX6-1+ PP cells, derived from different, both XY and XX, hPS cell lines, were enriched to nearly 90% homogeneity and expanded with very similar kinetics and efficiency. Furthermore, non-expanded and expanded PP cells, from different hPS cell lines, were differentiated in microwells into homogeneous islet-like clusters (SC-islets) with very similar efficiency. These clusters contained abundant β-cells of comparable functionality as assessed by glucose-stimulated insulin secretion assays. These findings established the signaling requirements to decouple PP proliferation from differentiation and allowed the consistent expansion of hPS cell-derived PP cells. They will enable the establishment of large banks of GMP-produced PP cells derived from diverse hPS cell lines. This approach will streamline SC-islet production for further development of the differentiation process, diabetes research, personalized medicine, and cell therapies.

Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions.

The unlimited expansion of human progenitor cells in vitro could unlock many prospects for regenerative medicine. However, it remains an important challenge as it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from those mechanisms promoting their differentiation. This study focuses on the expansion of human pluripotent stem (hPS) cell-derived pancreatic progenitors (PP) to advance novel therapies for diabetes. We obtained mechanistic insights into PP expansion requirements and identified conditions for the robust and unlimited expansion of hPS cell-derived PP cells under GMP-compliant conditions through a hypothesis-driven iterative approach. We show that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling, and inhibition of selected branches of the TGFβ and Wnt signaling pathways are necessary for the effective decoupling of PP proliferation from differentiation. This enabled the reproducible, 2000-fold, over 10 passages and 40-45 d, expansion of PDX1+/SOX9+/NKX6-1+ PP cells. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. Using these conditions, PDX1+/SOX9+/NKX6-1+ PP cells, derived from different, both XY and XX, hPS cell lines, were enriched to nearly 90% homogeneity and expanded with very similar kinetics and efficiency. Furthermore, non-expanded and expanded PP cells, from different hPS cell lines, were differentiated in microwells into homogeneous islet-like clusters (SC-islets) with very similar efficiency. These clusters contained abundant β-cells of comparable functionality as assessed by glucose-stimulated insulin secretion assays. These findings established the signaling requirements to decouple PP proliferation from differentiation and allowed the consistent expansion of hPS cell-derived PP cells. They will enable the establishment of large banks of GMP-produced PP cells derived from diverse hPS cell lines. This approach will streamline SC-islet production for further development of the differentiation process, diabetes research, personalized medicine, and cell therapies.

Stem cell culture and differentiation in 3-D scaffolds.

Conventional two-dimensional (2-D) cultivation are easy to utilize for human pluripotent stem (hPS) cell cultivation in standard techniques and are important for analysis or development of the signal pathways to keep pluripotent state of hPS cells cultivated on 2-D cell culture materials. However, the most efficient protocol to prepare hPS cells is the cell culture in a three dimensional (3-D) cultivation unit because huge numbers of hPS cells should be utilized in clinical treatment. Some 3-D cultivation strategies for hPS cells are considered: (a) microencapsulated cell cultivation in suspended hydrogels, (b) cell cultivation on microcarriers (MCs), (c) cell cultivation on self-aggregated spheroid [cell aggregates; embryoid bodies (EBs) and organoids], (d) cell cultivation on microfibers or nanofibers, and (e) cell cultivation in macroporous scaffolds. These cultivation ways are described in this chapter.

Universal and hypoimmunogenic pluripotent stem cells for clinical usage.

It is urgent to prepare and store large numbers of clinical trial grade human pluripotent stem (hPS) cells for off-the-shelf use in stem cell therapies. However, stem cell banks, which store off-the-shelf stem cells, need financial support and large amounts of technicians for daily cell maintenance. Therefore, it is valuable to create "universal" or "hypoimmunogenic" hPS cells with genome editing engineering by knocking in or out immune-related genes. Only a small number of universal or hypoimmunogenic hPS cell lines should be needed to store for off-the-shelf usage and reduce the large amounts of instruments, consumables and technicians. In this article, we consider how to create hypoimmunogenic or universal hPS cells as well as the demerits of the technology. β2-Microglobulin-knockout hPS cells did not harbor human leukocyte antigen (HLA)-expressing class I cells but led to the activation of natural killer cells. To escape the activities of macrophages and natural killer cells, homozygous hPS cells having a single allele of an HLA class I gene, such as HLA-C, were proposed. Major HLA class Ia molecules were knocked out, and CD47, HLA-G and PD-L1 were knocked in hPS cells utilizing CRISPR/Cas9 genome editing. Finally, some researchers are trying to generate universal hPS cells without genome editing. The cells evaded the activation of not only T cells but also macrophages and natural killer cells. These universal hPS cells have high potential for application in cell therapy.

The Hollow Porous Sphere Cell Carrier for the Dynamic Three-Dimensional Cell Culture.

Large-scale mammalian cell culture is essential in cell therapy, vaccine production, and the manufacturing of therapeutic protein drugs. Due to the adherent growth characteristic of most mammalian cell types, the combination of cell carrier and bioreactor is a common choice in large-scale mammalian cell culture. Current cell carriers developed by polymer crosslinking, lithography, or emulsion drops are unable to obtain a structure with uniformed porous structure and porous interior design, which results in an inhomogeneous culture condition for cells and therefore cannot ensure an optimal dynamic culture condition for cell proliferation, matrix production, and cell differentiation. In addition, the fluidic shear stress (a standard mechanical stimulation in bioreactor culture) and inner-carrier velocity (to ensure nutrient transport and waste exchange), which influence cell viability and growth, are not well-controlled/analyzed due to an irregular porous structure with these traditionally synthesized cell carriers. To solve these problems, we designed four types of hollow porous spheres (HPS, 1.0 cm diameter) with different porous structures. To investigate the impacts of porous structure on surface shear stress and inner velocity, computational fluid dynamics (CFD) simulations were conducted to analyze the liquid flow behavior in HPSs, based on which an optimal structure with minimal surface shear stress and best inner velocity was obtained and fabricated using fused deposition modeling three-dimensional (3D) printing technology. Inspired by the industrial large-scale culture system, a novel 3D dynamic culture system was then established using HPSs to seed the cells, which were then placed in a mini bioreactor on a tube roller. CFD analysis showed that under 0.1 m/s water flow, the shear stress at most surface areas from four HPSs was lower than 20 dynes/cm2, which suggests that the HPSs should provide protection against physical stress to the cells living on the scaffold surface. A dynamic cell seeding was developed and refined using the 3D culture system, which increased the 32% seeding efficiency of MC3T3 cells compared to the traditional static cell seeding method. The cell proliferation analysis demonstrated that HPSs could speed up cell growth in dynamic cell culture. The HPS with a honeycomb-like structure showed the highest inner pore velocity (CFD analysis) and achieved the fastest cell proliferation and the highest cell viability. Overall, our study, for the first time, developed a 3D printed HPS cell culture device with a uniformed porous structure, which can effectively facilitate cell adhesion and proliferation in the dynamic cultural environment, thereby could be considered an ideal carrier candidate.

Generation of Human Ventral Midbrain Organoids Derived from Pluripotent Stem Cells.

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide and is caused by the degeneration and loss of dopamine (DA) neurons in the ventral midbrain (VM). The focal and progressive degeneration of DA neurons in the VM makes PD a particularly attractive target for cell-based therapies. Human pluripotent stem cells (hPSCs) offer unprecedented opportunities to model the development and functional properties of human DA neurons in a dish. The use of human in vitro models based on hPSCs has empowered studies of VM development and provided access to neurons expressing a particular disease-specific phenotype. Currently, hPSC differentiation is most routinely carried out in monolayer cultures, which do not properly recapitulate cell-cell interactions and the structural complexity of the brain. Moreover, 2D cultures are challenging to maintain long term, as the cells tend to detach from the plate and lose their functional characteristics. This precludes the possibility of mimicking later phases of DA neurogenesis and recreating the complexity of functional neural circuitries. Here, we describe protocols showing how to maintain hPSCs in an undifferentiated state and how to then drive these hPSCs into 3D regionalized VM organoids. After long-term culture, these VM organoids exhibit mature and post-mitotic molecular features, including neuromelanin pigments similar to those released in primate VMs. We also report a protocol describing how to efficiently perform immunohistochemistry and how to detect neuromelanin-containing DA neurons in VM organoids. Together, these protocols provide a 3D in vitro platform that can be used to better understand the molecular mechanisms underlying DA neuron function and disease and may serve as a powerful tool for designing more targeted disease-modifying therapies. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Human pluripotent stem cell culture Basic Protocol 2: hPS cell differentiation for the generation of human ventral midbrain organoids Basic Protocol 3: Characterization of ventral midbrain organoids.

Innovation and issues in hPS cell bioprocessing

Innovation and issues in hPS cell bioprocessing

Clonal derivation of white and brown adipocyte progenitor cell lines from human pluripotent stem cells

BackgroundThe role of brown fat in non-shivering thermogenesis and the discovery of brown fat depots in adult humans has made it the subject of intense research interest. A renewable source of brown adipocyte (BA) progenitors would be highly valuable for research and therapy. Directed differentiation of human pluripotent stem (hPS) cells to white or brown adipocytes is limited by lack of cell purity and scalability. Here we describe an alternative approach involving the identification of clonal self-renewing human embryonic progenitor (hEP) cell lines following partial hPS cell differentiation and selection of scalable clones.MethodsWe screened a diverse panel of hPS cell-derived clonal hEP cell lines for adipocyte markers following growth in adipocyte differentiation medium. The transcriptome of the human hES-derived clonal embryonic progenitor cell lines E3, C4ELS5.1, NP88, and NP110 representing three class of definitive adipocyte progenitors were compared to the relatively non-adipogenic line E85 and adult-derived BAT and SAT-derived cells using gene expression microarrays, RT-qPCR, metabolic analysis and immunocytochemistry. Differentiation conditions were optimized for maximal UCP1 expression.ResultsMany of the differentiated hEP cell lines expressed the adipocyte marker, FAPB4, but only a small subset expressed definitive adipocyte markers including brown adipocyte marker, UCP1. Class I cells (i.e., E3) expressed CITED1, ADIPOQ, and C19orf80 but little to no UCP1. Class II (i.e., C4ELS5.1) expressed CITED1 and UCP1 but little ADIPOQ and LIPASIN. Class III (i.e., NP88, NP110) expressed CITED1, ADIPOQ, C19orf80, and UCP1 in a similar manner as fetal BAT-derived (fBAT) cells. Differentiated NP88 and NP110 lines were closest to fBAT cells morphologically in adiponectin and uncoupling protein expression. But they were more metabolically active than fBAT cells, had higher levels of 3-hydroxybutyrate, and lacked expression of fetal/adult marker, COX7A1. The hEP BA progenitor lines were scalable to 17 passages without loss of differentiation capacity and could be readily rederived.ConclusionsTaken together, these data demonstrate that self-renewing adipocyte progenitor cells can be derived from hES cells and that they are functionally like BAT cells but with unique properties that might be advantageous for basic research and for development of cell-based treatments for metabolic diseases.

MiR145-5p inhibits proliferation of PMVECs via PAI-1 in experimental hepatopulmonary syndrome rat pulmonary microvascular hyperplasia.

ABSTRACTHepatopulmonary syndrome (HPS) is a triad of advanced liver disease, intrapulmonary vasodilatation and arterial hypoxemia. Increasing evidence shows that HPS is associated with pulmonary microvascular hyperplasia. The aim of this work was to investigate the underlying mechanism of miR-145 in regulating the proliferation of pulmonary microvascular endothelial cells (PMVECs) and angiogenesis in HPS via plasminogen activator inhibitor-1 (PAI-1). To test this, morphology score and number of pulmonary microvascular were assessed in lung tissues from rats with HPS by Hematoxylin and Eosin (H&E) staining. Expression levels of PAI-1 were assessed in lung tissues from HPS rats, as well as in PMVECs treated with HPS rat serum. We also selected the putative microRNA binding site on PAI-1 by bioinformatics analysis. Then, miR145-3p and miR145-5p expression levels in the lungs and PMVECs of rats were detected by qRT-PCR because miR145-5p is a microRNA binding site on PAI-1. In addition, the effects of miR-145-5p regulation on PAI-1 were examined by upregulation and downregulation of miR-145-5p and specific lentivirus transfection was used to overexpress and knockdown PAI-1 to assess PAI-1 function on PMVECs proliferation. Our data showed that levels of PAI-1 expression in lung tissue of rats increased significantly when rats were treated with common bile duct ligation. We found that levels of miR-145-5p were frequently downregulated in HPS tissues and cell lines, and overexpression of miR-145-5p dramatically inhibited PMVECs proliferation. We further verified PAI-1 as a novel and direct target of miR-145-5p in HPS. MiR-145-5p inhibits PAI-1 synthesis and the expression changes of PAI-1 directly affect the proliferation of PMVECs. We concluded that miR-145-5p negatively regulates PMVEC proliferation through PAI-1 expression. In addition, overexpression of miR-145-5p may prove beneficial as a therapeutic strategy for HPS treatment.

Human Pluripotent Stem Cells: Applications and Challenges for Regenerative Medicine and Disease Modeling.

In recent years, human pluripotent stem (hPS) cells have started to emerge as a potential tool with application in fields such as regenerative medicine, disease modeling, and drug screening. In particular, the ability to differentiate human-induced pluripotent stem (hiPS) cells into different cell types and to mimic structures and functions of a specific target organ, resourcing to organoid technology, has introduced novel model systems for disease recapitulation while offering a powerful tool to provide a faster and reproducible approach in the process of drug discovery. All these technologies are expected to improve the overall quality of life of the humankind. Here, we highlight the main applications of hiPS cells and the main challenges associated with the translation of hPS cell derivatives into clinical settings and other biomedical applications, such as the costs of the process and the ability to mimic the complexity of the in vivo systems. Moreover, we focus on the bioprocessing approaches that can be applied towards the production of high numbers of cells as well as their efficient differentiation into the final product and further purification.

A new approach to measure the non-linear Butler–Volmer behavior of electrochemical systems in the time domain

A new approach to measure the non-linear Butler–Volmer behavior of electrochemical systems in the time domain is presented. The method is based on two superimposed currents, one large pulse as an offset and a small pulse which is transformed into the frequency domain. The advantage of this new method is the decreased measurement time compared to frequency domain measurements. This allows the characterization at lower frequencies with less distortion from change in SOC and temperature. In order to reduce spectral leakage of the fast Fourier transform the derivative of the measured signals is taken. The measurement time can be as small as the theoretical limit T=1/f with a negligible error. For the characterization of large automotive cells high currents are needed which were provided by a reprogrammed BaSyTeC HPS cell test system, also used to conduct dynamic electrochemical impedance spectroscopy measurements to validate the new method. The equality of the two completely different hardware and software systems is shown for a 50Ah cell for automotive applications at −20°C in a current range from 0 to 100A.

Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations.

Human pluripotent stem cells (hPSCs) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with acquisition of large copy number variants (CNVs) that provide mutant cells with a growth advantage in culture1–3. However, the nature, extent, and functional impact of other acquired genome sequence mutations in cultured hPSCs is not known. Here, we sequenced the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hESC) lines, including 26 lines prepared for potential clinical use4. We then applied computational strategies for identifying mutations present in a subset of cells5. Though such mosaic mutations were generally rare, we identified five unrelated hESC lines that carried six mutations in the TP53 gene that encodes the tumor suppressor P53. Notably, the TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We used droplet digital PCR to demonstrate that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that P53 mutation confers selective advantage. When we then mined published RNA sequencing data from 117 hPSC lines, we observed another nine TP53 mutations, all resulting in coding changes in the DNA binding domain of P53. Strikingly, in three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from loss of heterozygosity at the TP53 locus. As the acquisition and favored expansion of cancer-associated mutations in hPSCs may go unnoticed during most applications, we suggest that careful genetic characterization of hPSCs and their differentiated derivatives should be carried out prior to clinical use.

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture.

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications.

Multi-Scale Imaging and Informatics Pipeline for In Situ Pluripotent Stem Cell Analysis

Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However, hPS cells cultured in vitro exhibit a high degree of heterogeneity, presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures, necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution, with single-cellular and sub-cellular analysis at high resolutions, generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1, reflecting a less pluripotent state, while cells within the first pluripotent layer are more likely to be in G2, possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes, and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly, we demonstrate that our pipeline can robustly handle high-content, high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall, the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide, and more generally, multi-scale spatial configuration.

Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins.

The fate decisions of human pluripotent stem (hPS) cells are governed by soluble and insoluble signals from the microenvironment. Many hPS cell differentiation protocols use Matrigel, a complex and undefined substrate that engages multiple adhesion and signaling receptors. Using defined surfaces programmed to engage specific cell-surface ligands (i.e., glycosaminoglycans and integrins), the contribution of specific matrix signals can be dissected. For ectoderm and motor neuron differentiation, peptide-modified surfaces that can engage both glycosaminoglycans and integrins are effective. In contrast, surfaces that interact selectively with glycosaminoglycans are superior to Matrigel in promoting hPS cell differentiation to definitive endoderm and mesoderm. The modular surfaces were used to elucidate the signaling pathways underlying these differences. Matrigel promotes integrin signaling, which in turn inhibits mesendoderm differentiation. The data indicate that integrin-activating surfaces stimulate Akt signaling via integrin-linked kinase (ILK), which is antagonistic to endoderm differentiation. The ability to attribute cellular responses to specific interactions between the cell and the substrate offers new opportunities for revealing and controlling the pathways governing cell fate.

Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification.

Physical stimuli can act in either a synergistic or antagonistic manner to regulate cell fate decisions, but it is less clear whether insoluble signals alone can direct human pluripotent stem (hPS) cell differentiation into specialized cell types. We previously reported that stiff materials promote nuclear localization of the Yes-associated protein (YAP) transcriptional coactivator and support long-term self-renewal of hPS cells. Here, we show that even in the presence of soluble pluripotency factors, compliant substrata inhibit the nuclear localization of YAP and promote highly efficient differentiation of hPS cells into postmitotic neurons. In the absence of neurogenic factors, the effective substrata produce neurons rapidly (2 wk) and more efficiently (>75%) than conventional differentiation methods. The neurons derived from substrate induction express mature markers and possess action potentials. The hPS differentiation observed on compliant surfaces could be recapitulated on stiff surfaces by adding small-molecule inhibitors of F-actin polymerization or by depleting YAP. These studies reveal that the matrix alone can mediate differentiation of hPS cells into a mature cell type, independent of soluble inductive factors. That mechanical cues can override soluble signals suggests that their contributions to early tissue development and lineage commitment are profound.

SPARC is involved in the maintenance of mitotically inactivated mouse embryonic fibroblast cells

Mitotically inactivated feeder cells such as mouse embryonic fibroblast (MEFs) cells have been widely applied for physical and physiological support in the pluripotency maintenance of human pluripotent stem cells (hPSCs). However, accurate supporting mechanism or factors of feeder cells are poorly understood. Here, we isolated differentially expressed genes between wild-type MEFs and mitotically inactivated MEFs (miMEFs) by employing annealing control primer-based GeneFishing polymerase chain reaction. We identified a secreted protein acidic cysteine-rich glycoprotein (SPARC) gene that is upregulated in miMEFs. Suppression of SPARC expression in miMEFs using small interference RNA (siRNA) displayed gradual detachment of miMEFs. Furthermore, we found a significant reduction of OCT4- and SSEA3-positive hPS cell population maintained on SPARC siRNA-miMEFs compared to on miMEFs by flow cytometrical analysis. These findings suggest that SPARC plays a critical role in the maintenance of miMEFs without loss of cell number and might be a key component for supporting the culture of hPSCs.