• Samsung Research Funding Center of Samsung Electronics
• National Research Foundation of Korea

• Cell adhesion molecules
• DPPA3
• Induced pluripotent stem cells
• Integrin
• Reprogramming

• biomaterials
• biomimetic
• extracellular matrices
• hydrogels
• laminins
• tissue engineering

• Hydrogels/chemistry
• Hydrogels/pharmacology
• Laminin/chemistry
• Laminin/pharmacology
• Tissue Engineering/methods
• Humans
• Biocompatible Materials/chemistry
• Biocompatible Materials/pharmacology
• Animals
• Extracellular Matrix/chemistry
• Peptides/chemistry
• Peptides/pharmacology

• SPG/2020/000748 SERB

• Akt
• Oct4
• Rac1
• basement membrane
• human embryogenesis
• human embryoid

• Humans
• Basement Membrane/metabolism
• Embryonic Development/genetics
• Octamer Transcription Factor-3/metabolism
• Octamer Transcription Factor-3/genetics
• rac1 GTP-Binding Protein/metabolism
• rac1 GTP-Binding Protein/genetics
• Signal Transduction
• Proto-Oncogene Proteins c-akt/metabolism
• Gene Expression Regulation, Developmental
• Primitive Streak/metabolism
• Primitive Streak/cytology
• Laminin/metabolism
• Extracellular Matrix/metabolism

• Chromatin remodelling
• Epigenetic regulation
• Mechanobiology
• Mechanotransduction
• Multipotent stem cells
• Pluripotent stem cells

• Mechanotransduction, Cellular
• Humans
• Animals
• Stem Cells/metabolism
• Stem Cells/cytology
• Cell Differentiation

• canine induced pluripotent stem cell
• culture system
• differentiation propensity
• feeder-free
• weekend-free

• Animals
• Dogs
• Humans
• Induced Pluripotent Stem Cells/metabolism
• Pluripotent Stem Cells/metabolism
• Cell Differentiation
• Embryoid Bodies

• FGFR
• Human pluripotent stem cells
• Integrin
• Maintenance of pluripotency
• bFGF

• PMEDSAH
• human embryonic stem cells
• human pluripotent stem cells
• reprogramming
• self-renewal
• synthetic polymer grafting

• 1828253, 2026049 and 2234541. NSF

• Basement membrane
• Ion channels
• Laminin
• Myoendothelial junctions
• Resistance arteries

• Mice
• Humans
• Animals
• Laminin/genetics
• Laminin/metabolism
• Endothelial Cells/metabolism
• TRPV Cation Channels/genetics
• TRPV Cation Channels/metabolism
• Basement Membrane/metabolism
• Endothelium, Vascular/metabolism
• Communication

• Biological sciences
• Immune response
• Immune system
• Immunology

• Biomaterial
• Cell cultivation
• Cell differentiation
• Embryonic stem cells
• Extracellular matrix
• Hydrogel
• Induced pluripotent stem cells
• Peptide

• Animals
• Mice
• Adult
• Humans
• Laminin/pharmacology
• Fibroblasts
• Adult Stem Cells
• Biocompatible Materials/pharmacology
• Cell Proliferation

• cardiac tissue engineering
• cardiomyocytes
• developmental biology
• differentiation
• extracellular matrix
• hESC
• heart models
• human embryonic stem cells
• iPSC
• induced pluripotent stem cells

• cardiomyocyte
• coating material
• human induced pluripotent stem cells (hiPSCs)
• regenerative medicine
• therapeutic cells

• Japan Agency for Medical Research and Development

• chemically defined
• human pluripotent stem cells
• peptide
• synthetic surface
• three-dimensional culture

• Expansion
• Nanocasting
• Substrate
• hiPSC

• embryonic stem cell (ES cells)
• heart failure
• induced pluripotent stem cell (iPS cell) (iPSC)
• regenerative therapy
• stem cell metabolism

Human amniotic cells (hAC) exhibit characteristics of undifferentiated cells and immunomodulatory properties. Recognition of the relationship between amniotic cells and components of the extracellular matrix is an important condition for their ex vivo preparation and further successful clinical application in regenerative medicine and transplantology. Laminin 332 (LN-332), as a natural component of the basement membrane of amniotic epithelial cells and a ligand for integrin receptors, may strongly influence the phenotype and fate of amniotic cells. We investigated the impact of recombinant LN-332 on hAC viability and expression of markers for pluripotency, early differentiation, adhesion, and immunomodulatory properties. During 14 days of culture, hAC were quantified and qualified by light microscopy, immunohistochemistry, immunocytochemistry, and flow cytometry. Gene expression was assessed with real-time polymerase chain reaction (RT-PCR) arrays and compared with differentiated cells originated from the three germ layers. LN-332 caused an over 2-fold increase in the total number of hAC, accompanied by a 75% reduction of SSEA-4-positive cells and an increase in HLA-ABC-positive cells. In particular, we observed that the presence of laminin 332 in the medium of a short-time culture modifies the effect of culture duration on hAC, enhancing time-dependent inhibition of expression of certain genes, including pluripotency and differentiation markers, laminin 332 subunits (which may be part of self-regulation of LN-332 synthesis by amniotic cells), and integrins. The changes observed in hAC were more distinct with respect to differentiated mesenchymal cells, resulting in more comparable phenotypes than those represented by differentiated endo- and ectodermal cells. We concluded that laminin 332 present in the culture medium influences to a certain extent proliferation, adhesion, and differentiation of amniotic cells in culture.

• basement membrane
• extracellular matrix
• gene expression
• human amniotic cells
• in vitro cell differentiation
• integrins
• laminin 332
• pluripotency

• high-throughput microculture
• human pluripotent stem cells
• laminin-mimetic
• peptide self-assembled monolayers
• targeted peptide
• α6-integrin

• Atmospheric pressure plasma
• Embryonic stem cell
• Surface modification
• UV/ozone surface modification
• X-ray photoelectron spectroscopy

Over the last century, diabetes has been treated with subcutaneous insulin, a discovery that enabled patients to forego death from hyperglycemia. Despite novel insulin formulations, patients with diabetes continue to suffer morbidity and mortality with unsustainable costs to the health care system. Continuous glucose monitoring, wearable insulin pumps, and closed-loop artificial pancreas systems represent an advance, but still fail to recreate physiologic euglycemia and are not universally available. Islet cell transplantation has evolved into a successful modality for treating a subset of patients with ‘brittle’ diabetes but is limited by organ donor supply and immunosuppression requirements. A novel approach involves generating autologous or immune-protected islet cells for transplant from inducible pluripotent stem cells to eliminate detrimental immune responses and organ supply limitations. In this review, we briefly discuss novel mechanisms for subcutaneous insulin delivery and define their shortfalls. We describe embryological development and physiology of islets to better understand their role in glycemic control and, finally, discuss cell-based therapies for diabetes and barriers to widespread use. In response to these barriers, we present the promise of stem cell therapy, and review the current gaps requiring solutions to enable widespread use of stem cells as a potential cure for diabetes.

• diabetes
• immune reset
• immunosuppression
• inducible pluripotent stem cells
• insulin
• islet cell transplant

• Cell- and Tissue-Based Therapy/methods
• Diabetes Mellitus, Type 1/therapy
• Humans
• Induced Pluripotent Stem Cells/metabolism
• Islets of Langerhans Transplantation/methods

Solid cancers are surrounded by a network of non-cancerous cells comprising different cell types, including fibroblasts, and acellular protein structures. This entire network is called the tumor microenvironment (TME) and it provides a physical barrier to the tumor shielding it from infiltrating immune cells, such as lymphocytes, or therapeutic agents. In addition, the TME has been shown to dampen efficient immune responses of infiltrated immune cells, which are key in eliminating cancer cells from the organism. In this review, we will discuss how TME proteins in particular are involved in this dampening effect, known as immunosuppression. We will focus on three different types of digestive cancers: pancreatic cancer, colorectal cancer, and gastric cancer. Moreover, we will discuss current therapeutic approaches using TME proteins as targets to reverse their immunosuppressive effects.

The stromal tumor microenvironment (TME) consists of immune cells, vascular and neural structures, cancer-associated fibroblasts (CAFs), as well as extracellular matrix (ECM), and favors immune escape mechanisms promoting the initiation and progression of digestive cancers. Numerous ECM proteins released by stromal and tumor cells are crucial in providing physical rigidity to the TME, though they are also key regulators of the immune response against cancer cells by interacting directly with immune cells or engaging with immune regulatory molecules. Here, we discuss current knowledge of stromal proteins in digestive cancers including pancreatic cancer, colorectal cancer, and gastric cancer, focusing on their functions in inhibiting tumor immunity and enabling drug resistance. Moreover, we will discuss the implication of stromal proteins as therapeutic targets to unleash efficient immunotherapy-based treatments.

• digestive cancers
• extracellular matrix (ECM) proteins
• immune regulation
• tumor microenvironment (TME)

• Cell culture
• Cryopreservation
• Human induced pluripotent stem cells
• Pluripotency

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for the creation of cartilage to treat articular cartilage damage. The molecular mechanisms that translate culture conditions to the chondrogenic differentiation of hiPSCs remain to be analyzed. To analyze the effects of culture substrates, we chondrogenically differentiated hiPSCs on Matrigel or laminin 511‐E8 while holding the composition of the chondrogenic medium constant. Cartilage was formed from hiPSCs on Matrigel, but not on laminin 511‐E8. On Matrigel, the hiPSCs were round and yes‐associated protein (YAP) was inactive. In contrast, on laminin 511‐E8, the hiPSCs were flat and YAP was active. Treating the laminin 511‐E8 hiPSCs in a bioreactor caused cell aggregates, in which the cells were round and YAP was inactive. Subsequent culture of the aggregates in chondrogenic medium resulted in cartilage formation. Transient knockdown of YAP in hiPSCs around the start of chondrogenic differentiation successfully formed cartilage on laminin 511‐E8, suggesting that the activation of YAP is responsible for the failure of cartilage formation from hiPSCs on laminin 511‐E8. Consistently, the addition of YAP inhibitors to laminin 511‐E8 hiPSCs caused partial cartilage formation. This study contributes to identifying the molecules that mediate the effects of culture substrates on the chondrogenic differentiation of hiPSCs as well as to developing clinically applicable chondrogenic differentiation methods.

• cartilage
• chondrocyte
• chondrogenesis
• differentiation
• iPSCs
• pluripotent stem cells
• stem cell culture

Pluripotent stem cells (PSCs) are considered as being an important cell source for regenerative medicine. The culture of PSCs usually requires a feeder cell layer or cell adhesive matrix coating such as Matrigel, laminin, and gelatin. Although a feeder-free culture using a matrix coating has been popular, the on-feeder culture is still an effective method for the fundamental study of regenerative medicine and stem cell biology. To culture PSCs on feeder cell layers, the elimination of feeder cells is required for biological or gene analysis and for cell passage. Therefore, a simple and cost-effective cell sorting technology is required. There are several commercialized cell-sorting methods, such as FACS or MACS. However, these methods require cell labeling by fluorescent dye or magnetic antibodies with complicated processes. To resolve these problems, we focused on dielectrophoresis (DEP) phenomena for cell separation because these do not require any fluorescent or magnetic dyes or antibodies. DEP imposes an electric force on living cells under a non-uniform AC electric field. The direction and magnitude of the DEP force depend on the electric property and size of the cell. Therefore, DEP is considered as a promising approach for sorting PSCs from feeder cells. In this study, we developed a simple continuous cell-sorting device using the DEP force and fluid-induced shear force. As a result, mouse embryonic stem cells (mESCs) were purified from a mixed-cell suspension containing mESCs and mouse embryonic fibroblasts (MEFs) using our DEP cell-sorting device.

• cell sorting
• dielectrophoresis
• feeder cell
• fluid-induced shear force
• pluripotent stem cell

• R01 HL093282 NHLBI NIH HHS
• R01 NS109427 NINDS NIH HHS
• T32 HL110853 NHLBI NIH HHS
• U01 TR002383 NCATS NIH HHS

• OSKM
• clinical applications
• gene editing
• iPSCs
• reprogramming
• viral and nonviral vectors

• Cell adhesive matrix
• Cell culture plastics
• Human induced pluripotent stem cell
• Surface chemistry
• UV/ozone surface modification

• cell therapy
• clinical manufacturing
• induced pluripotent stem cells
• quality control

Human induced pluripotent stem cells (hiPSCs) grow indefinitely in culture and have the potential to regenerate various tissues. In the development of cell culture systems, a fragment of laminin-511 (LM511-E8) was found to improve the proliferation of stem cells. The adhesion of undifferentiated cells to LM511-E8 is mainly mediated through integrin α6β1. However, the involvement of non-integrin receptors remains unknown in stem cell culture using LM511-E8. Here, we show that dystroglycan (DG) is strongly expressed in hiPSCs. The fully glycosylated DG is functionally active for laminin binding, and although it has been suggested that LM511-E8 lacks DG binding sites, the fragment does weakly bind to DG. We further identified the DG binding sequence in LM511-E8, using synthetic peptides, of which, hE8A5-20 (human laminin α5 2688–2699: KTLPQLLAKLSI) derived from the laminin coiled-coil domain, exhibited DG binding affinity and cell adhesion activity. Deletion and mutation studies show that LLAKLSI is the active core sequence of hE8A5-20, and that, K2696 is a critical amino acid for DG binding. We further demonstrated that hiPSCs adhere to hE8A5-20-conjugated chitosan matrices. The amino acid sequence of DG binding peptides would be useful to design substrata for culture system of undifferentiated and differentiated stem cells.

The enteric glia, a neural crest-derived cell type that composes the Enteric Nervous System, is involved in controlling gut functions, including motility, gut permeability, and neuronal communication. Moreover this glial cell could to give rise to new neurons. It is believed that enteric neurons are generated up to 21 days postnatally; however, adult gut cells with glial characteristics can give rise to new enteric neurons under certain conditions. The factors that activate this capability of enteric glia to differentiate into neurons remain unknown. Here, we followed the progress of this neuronal differentiation and investigated this ability by challenging enteric glial cells with different culture conditions. We found that, in vitro, enteric glial cells from the gut of adult and neonate mice have a high capability to acquire neuronal markers and undergoing morphological changes. In a co-culture system with 3T3 fibroblasts, the number of glial cells expressing βIIItubulin decreased after 7 days. The effect of 3T3-conditioned medium on adult cells was not significant, and fewer enteric glial cells from neonate mice began the neurogenic process in this medium. Laminin, an extracellular matrix protein that is highly expressed by the niche of the enteric ganglia, seemed to have a large role in inhibiting the differentiation of enteric glia, at least in cells from the adult gut. Our results suggest that, in an in vitro approach that provides conditions more similar to those of enteric glial cells in vivo, these cells could, to some extent, retain their morphology and marker expression, with their neurogenic potential inhibited. Importantly, laminin seemed to inhibit differentiation of adult enteric glial cells. It is possible that the differentiation of enteric glia into neurons is related to severe changes in the microenvironment, leading to disruption of the basement membrane. In summary, our data indicated that the interaction between the enteric glial cells and their microenvironment molecules significantly affects the control of their behavior and functions.

• cell culture
• enteric glia
• laminin
• microenvironment
• neurogenesis

• E‐cadherin
• Wnt signaling pathway
• embryonic stem cells
• pluripotency
• reduced graphene oxide

• cardiomyocyte differentiation
• expansion culture
• human pluripotent stem cells (hPSCs)

• BM, Basement membrane
• Basement membrane
• Cell–extracellular matrix interaction
• ECM, Extracellular matrix
• Epiblast stem cell
• Post-implantation embryo

• cell culture
• cell therapy
• defined substrates
• human pluripotent stem cells
• human recombinant laminins

Conventional cell handling and sorting methods require manual labor, which decreases both cell quality and quantity. To purify adherent cultured cells, cell purification technologies that are high throughput without dissociation and can be utilized in an on-demand manner are expected. Here, we developed a Laser-induced, Light-responsive-polymer-Activated, Cell Killing (LiLACK) system that enables high-speed and on-demand adherent cell sectioning and purification. This system employs a visible laser beam, which does not kill cells directly, but induces local heat production through the trans-cis-trans photo-isomerization of azobenzene moieties. Using this system in each passage for sectioning, human induced pluripotent stem cells (hiPSCs) maintained their pluripotency and self-renewal during long-term culture. Furthermore, combined with deep machine-learning analysis on fluorescent and phase contrast images, a label-free and automatic cell processing system has been developed by eliminating unwanted spontaneously differentiated cells in undifferentiated hiPSC culture conditions.

Yohei Hayashi et al. present a method for high-speed adherent cell sectioning and purification, along with a label-free and automatic cell processing system. They show that this method is able to section human induced pluripotent stem cells without losing pluripotency and viability.

Understanding how extracellular matrix (ECM) protein composition regulates the process of human induced pluripotent stem cell (hiPSC) colony formation may facilitate the design of optimal cell culture environments. In this study, we investigated the effect of migratory behaviors on hiPSC colony formation on various ECM-coated surfaces.

To quantify how different ECM proteins affect migratory behavior during the colony formation process, single cells were seeded onto surfaces coated with varying concentrations of different ECM proteins. Cell behavior was monitored by time-lapse observation, and quantitative analysis of migration rates in relation to colony formation patterns was performed. Actin cytoskeleton, focal adhesions, and cell–cell interactions were detected by fluorescence microscopy.

Time-lapse observations revealed that different mechanisms of colony formation were dependent upon the migratory behavior of cells on different ECM surfaces. HiPSCs formed tight colonies on concentrated ECM substrates, while coating with dilute concentrations of ECM yielded more motile cells and colonies capable of splitting into single cells or small clusters. Enhanced migration caused a reduction of cell–cell contacts that enabled splitting or merging between cells and cell clusters, consequently reducing the efficiency of clonal colony formation. High cell-to-cell variability in migration responses to ECM surfaces elicited differential focal adhesion formation and E-cadherin expression within cells and colonies. This resulted in variability within focal adhesions and further loss of E-cadherin expression by hiPSCs.

Migration is an important factor affecting hiPSC colony-forming patterns. Regulation of migratory behavior can be an effective way to improve the expansion of hiPSCs while improving the process of clonal colony formation. We believe that this investigation provides a valuable method for understanding cell phenotypes and heterogeneity during colony formation in culture.

•

hiPSC colony-forming patterns were dependent on migratory behavior on different ECM surfaces.

• Cell migration
• Cell–cell interaction
• Clonal colony formation
• Cytoskeletal formation
• Extracellular matrix
• Human induced pluripotent stem cells

• Chitosan
• FGF-2
• Immobilization
• Induced pluripotent stem cells
• Vitronectin

• coacervation
• glycosaminoglycans
• material screening
• peptides
• stem cells

• Cell Culture Techniques
• Cell Differentiation
• Cells, Cultured
• Extracellular Matrix
• Mesenchymal Stem Cells
• Neural Stem Cells
• Osteogenesis

Human embryonic stem (hES) cells represent an important tool to study early cell development. The previously described use of human recombinant laminin (LN) 521 represented a step forward in generating clinically safe culture conditions. To test the short-term effect of LN521 on cultured hES cells, five male hES cell lines were cultured on human foreskin fibroblasts (hFFs), Matrigel, LN521, and LN121 and characterized by qPCR, immunofluorescence analysis, as well as their potential for three-germ layer differentiation. Variations in gene expression related to pluripotency, stemness, and testicular cells at different passages and culture conditions were evaluated by qPCR. All cell lines expressed pluripotency markers at protein and RNA level and were able to differentiate into cell types of the three germ layers after being cultured on LN521 for nine passages. Reduction in variation of pluripotency marker expression could be observed after culturing the cells on LN521 for nine passages. hES cells cultured on LN521 exhibited less differentiation, faster cell growth, and attachment when compared to hES cells cultured on LN121 or Matrigel. Our results indicate a positive effect of LN521 in stabilizing pluripotency gene expression and might be the first step towards more controllable and robust culture conditions for hES cells.