RESUMEN
The remarkable success of two FDA-approved mRNA-encapsulating vaccines (Comirnaty® and Spikevax®) indicated the importance of lipid nanoparticles (LNPs) delivery systems in clinical use. Currently, mRNA-encapsulating LNPs (mRNA-LNPs) vaccines are stored as frozen liquid at low or ultralow temperatures. We designed lyophilized LNPs utilizing FDA-approved lipids to expedite the clinical application of our developed lyophilized mRNA-LNPs in the future. The key parameters of sucrose concentration and the selection and molar ratio of the four lipids in these vaccines were optimized for long-term stability with high transfection efficiency after lyophilization. We demonstrated that 8.7% sucrose is the optimal cryoprotectant concentration to maintain the transfection efficiency of lyophilized mRNA-LNPs. Optimal lipid formulations with high transfection efficiency both before and after lyophilization were screened using an orthogonal experimental design. The ratios of distearoylphosphatidylcholine (DSPC)/cholesterol and the selection of the ionizable and PEGylated lipids are the main factors influencing the long-term stability of mRNA-LNPs. Comparative mouse transfection experiments showed that the optimal lyophilized mRNA-LNPs maintained high mRNA expression after lyophilization, predominantly in the spleen or liver, with no expression in the kidneys or eyes. Our studies demonstrated the importance of the sucrose concentration and of the selection and molar ratio of the four lipids composing LNPs for maintaining mRNA-LNP stability under lyophilization and for long-term storage under mild conditions.
RESUMEN
Millions of people worldwide have hereditary genetic disorders, trauma, infectious diseases, or cancer of the eyes, and many of these eye diseases lead to irreversible blindness, which is a major public health burden. The eye is a relatively small and immune-privileged organ. The use of nucleic acid-based drugs to manipulate malfunctioning genes that target the root of ocular diseases is regarded as a therapeutic approach with great promise. However, there are still some challenges for utilizing nucleic acid therapeutics in vivo because of certain unfavorable characteristics, such as instability, biological carrier-dependent cellular uptake, short pharmacokinetic profiles in vivo (RNA), and on-target and off-target side effects (DNA). The development of lipid nanoparticles (LNPs) as gene vehicles is revolutionary progress that has contributed the clinical application of nucleic acid therapeutics. LNPs have the capability to entrap and transport various genetic materials such as small interfering RNA, mRNA, DNA, and gene editing complexes. This opens up avenues for addressing ocular diseases through the suppression of pathogenic genes, the expression of therapeutic proteins, or the correction of genetic defects. Here, we delve into the cutting-edge LNP technology for ocular gene therapy, encompassing formulation designs, preclinical development, and clinical translation.
RESUMEN
Completely synthetic cell cultivation materials for human pluripotent stem cells (hPSCs) are important for the future clinical use of hPSC-derived cells. Currently, cell culture materials conjugated with extracellular matrix (ECM)-derived peptides are being prepared using only one specific integrin-targeting peptide. We designed dual peptide-conjugated hydrogels, for which each peptide was selected from different ECM sites: the laminin ß4 chain and fibronectin or vitronectin, which can target α6ß1 and α2ß1 or αVß5. hPSCs cultured on dual peptide-conjugated hydrogels, especially on hydrogels conjugated with peptides obtained from the laminin ß4 chain and vitronectin with a low peptide concentration of 200 µg/mL, showed high proliferation ability over the long term and differentiated into cells originating from 3 germ layers in vivo as well as a specific lineage of cardiac cells. The design of grafting peptides was also important, for which a joint segment and positive amino acids were added into the designed peptide. Because of the designed peptides on the hydrogels, only 200 µg/mL peptide solution was sufficient for grafting on the hydrogels, and the hydrogels supported hPSC cultures long-term; in contrast, in previous studies, greater than 1000 µg/mL peptide solution was needed for the grafting of peptides on cell culture materials.
RESUMEN
BACKGROUND: Retinal degeneration (RD) is a group of disorders on irreversible vision loss. Multiple types of stem cells were used in clinical trials for RD treatment. However, it remains unknown what kinds of stem cells are most effective for the treatment. Therefore, we investigated the subretinal transplantation of several types of stem cells, human adipose-derived stem cells (hADSCs), amniotic fluid stem cells (hAFSCs), bone marrow stem cells (hBMSCs), dental pulp stem cells (hDPSCs), induced pluripotent stem cell (hiPSC), and hiPSC-derived retinal pigment epithelium (RPE) cells for protection effects, paracrine effects and treatment efficiency in an RD disease model rats. METHODS: The generation and characterization of these stem cells and hiPSC-derived RPE cells were performed before transplantation. The stem cells or hiPSC-derived RPE cell suspension labelled with CellTracker Green to detect transplanted cells were delivered into the subretinal space of 3-week-old RCS rats. The control group received subretinal PBS injection or non-injection. A series of detections including fundus photography, optomotor response (OMR) evaluations, light-dark box testing, electroretinography (ERG), and hematoxylin and eosin (HE) staining of retinal sections were conducted after subretinal injection of the cells. RESULTS: Each stem cell, hiPSC-derived RPE cell or PBS (blank experiment) was successfully transplanted into at least six RCS rats subretinally. Compared with the control rats, RCS rats subjected to subretinal transplantation of any stem cells except hiPSCs showed higher ERG waves (p < 0.05) and quantitative OMR (qOMR) index values (hADSCs: 1.166, hAFSCs: 1.249, hBMSCs: 1.098, hDPSCs: 1.238, hiPSCs: 1.208, hiPSC-RPE cells: 1.294, non-injection: 1.03, PBS: 1.06), which indicated better visual function, at 4 weeks post-injection. However, only rats that received hiPSC-derived RPE cells maintained their visual function at 8 weeks post-injection (p < 0.05). The outer nuclear layer thickness observed in histological sections after HE staining showed the same pattern as the ERG and qOMR results. CONCLUSIONS: Compared to hiPSC-derived RPE cells, adult and fetal stem cells yielded improvements in visual function for up to 4 weeks post-injection; this outcome was mainly based on the paracrine effects of several types of growth factors secreted by the stem cells. Patients with RD will benefit from the stem cell therapy.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Mesenquimatosas , Degeneración Retiniana , Adulto , Humanos , Ratas , Animales , Degeneración Retiniana/terapia , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Retina/patología , Electrorretinografía , Células Madre Mesenquimatosas/metabolismo , Epitelio Pigmentado de la Retina/patologíaRESUMEN
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.
Asunto(s)
Técnicas de Cultivo Tridimensional de Células , Diferenciación Celular , Células Madre , Humanos , Células Madre/citología , Técnicas de Cultivo de Célula , Técnicas de Cultivo Tridimensional de Células/métodos , Hepatocitos/citología , Hidrogeles , Encapsulación Celular , Andamios del Tejido , Ingeniería de TejidosRESUMEN
Macular degeneration (MD) is a group of diseases characterized by irreversible and progressive vision loss. Patients with MD suffer from severely impaired central vision, especially elderly people. Currently, only one type of MD, wet age-related macular degeneration (AMD), can be treated with anti-vascular endothelium growth factor (VEGF) drugs. Other types of MD remain difficult to treat. With the advent of human pluripotent stem cells (hPSCs) and their differentiation into retinal pigmented epithelium (RPE), it is promising to treat patients with MD by transplantation of hPSC-derived RPE into the subretinal space. In this review, the current progress in hPSC-derived RPE transplantation for the treatment of patients with MD is described from bench to bedside, including hPSC differentiation into RPE and the characterization and usage of hPSC-derived RPE for transplantation into patients with MD.
Asunto(s)
Degeneración Macular , Células Madre Pluripotentes , Anciano , Humanos , Degeneración Macular/terapia , Diferenciación CelularRESUMEN
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. ß2-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.
Asunto(s)
Células Madre Pluripotentes , Trasplante de Células Madre , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/inmunología , Células Madre Pluripotentes/metabolismo , Antígenos HLA , Humanos , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Edición Génica , Técnicas de Sustitución del Gen , Animales , Inmunología del Trasplante , Bancos de Muestras BiológicasRESUMEN
Stem cells have self-renewal capability and can proliferate and differentiate into a variety of functionally active cells that can serve in various tissues and organs. This review discusses the history, definition, and classification of stem cells. Human pluripotent stem cells (hPSCs) mainly include embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Embryonic stem cells are derived from the inner cell mass of the embryo. Induced pluripotent stem cells are derived from reprogramming somatic cells. Pluripotent stem cells have the ability to differentiate into cells derived from all three germ layers (endoderm, mesoderm, and ectoderm). Adult stem cells can be multipotent or unipotent and can produce tissue-specific terminally differentiated cells. Stem cells can be used in cell therapy to replace and regenerate damaged tissues or organs.
Asunto(s)
Células Madre Adultas , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Adulto , Humanos , Células Madre Embrionarias , Diferenciación CelularRESUMEN
Human pluripotent stem cells (human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs)) have unlimited proliferative potential, whereas adult stem cells such as bone marrow-derived stem cells and adipose-derived stem cells have problems with aging. When hPSCs are intended to be cultured on feeder-free or xeno-free conditions without utilizing mouse embryonic fibroblasts or human fibroblasts, they cannot be cultured on conventional tissue culture polystyrene dishes, as adult stem cells can be cultured but should be cultivated on material surfaces grafted or coated with (a) natural or recombinant extracellular matrix (ECM) proteins, (b) ECM protein-derived peptides and specific synthetic polymer surfaces in xeno-free and/or chemically defined conditions. This review describes current developing cell culture biomaterials for the proliferation of hPSCs while maintaining the pluripotency and differentiation potential of the cells into 3 germ layers. Biomaterials for the cultivation of hPSCs without utilizing a feeder layer are essential to decrease the risk of xenogenic molecules, which contributes to the potential clinical usage of hPSCs. ECM proteins such as human recombinant vitronectin, laminin-511 and laminin-521 have been utilized instead of Matrigel for the feeder-free cultivation of hPSCs. The following biomaterials are also discussed for hPSC cultivation: (a) decellularized ECM, (b) peptide-grafted biomaterials derived from ECM proteins, (c) recombinant E-cadherin-coated surface, (d) polysaccharide-immobilized surface, (e) synthetic polymer surfaces with and without bioactive sites, (f) thermoresponsive polymer surfaces with and without bioactive sites, and (g) synthetic microfibrous scaffolds.
Asunto(s)
Células Madre Adultas , Laminina , Animales , Ratones , Adulto , Humanos , Laminina/farmacología , Fibroblastos , Materiales Biocompatibles/farmacología , Proliferación CelularRESUMEN
RNA, including mRNA, siRNA and miRNA, is part of a new class of patient treatments that prevent and treat several diseases. As an alternative to DNA therapy using plasmid DNA, RNA functions in the cellular cytosol, avoiding the potential risks of insertion into patient genomes. RNA drugs, including mRNA vaccines, need carrier materials for delivery into the patient's body. Several delivery carriers of mRNA, such as cationic polymers, lipoplexes, lipid-polymer nanoparticles and lipid nanoparticles (LNPs), have been investigated. For clinical applications, one of the most commonly selected types of RNA delivery carrier is LNPs, which are typically formed with (a) ionizable lipids, which bind to RNA; (b) cholesterol for stabilization; (c) phospholipids to form the LNPs; and (d) polyethylene glycol-conjugated lipids to prevent aggregation and provide stealth characteristics. Most RNA-LNP research has been devoted to achieving highly efficient RNA expression in vitro and in vivo. It is also necessary to study the extended storage of RNA-LNPs under mild conditions. One of the most efficient methods to store RNA-LNPs for a long time is to prepare freeze-dried (lyophilized) RNA-LNPs. Future research should include investigating LNP materials for the development of freeze-dried RNA-LNPs using optimal lipid components and compositions with optimal cryoprotectants. Furthermore, the development of sophisticated RNA-LNP materials for targeted transfection into specific tissues, organs or cells will be a future direction in the development RNA therapeutics. We will discuss the prospects for the development of next-generation RNA-LNP materials.
Asunto(s)
Lípidos , Nanopartículas , Humanos , Transfección , ARN Interferente Pequeño , ARN Mensajero/genética , LiofilizaciónRESUMEN
Human cells, especially stem cells, need to communicate and interact with extracellular matrix (ECM) proteins, which not only serve as structural components but also guide and support cell fate and properties such as cell adhesion, proliferation, survival and differentiation. The binding of the cells with ECM proteins or ECM-derived peptides via cell adhesion receptors such as integrins activates several signaling pathways that determine the cell fate, morphological change, proliferation and differentiation. The development of synthetic ECM protein-derived peptides that mimic the biological and biochemical functions of natural ECM proteins will benefit academic and clinical application. Peptides derived from or inspired by specific ECM proteins can act as agonists of each ECM protein receptor. Given that most ECM proteins function in cell adhesion via integrin receptors, many peptides have been developed that bind to specific integrin receptors. In this review, we discuss the peptide sequence, immobilization design, reaction method, and functions of several ECM protein-derived peptides. Various peptide sequences derived from mainly ECM proteins, which are used for coating or grafting on dishes, scaffolds, hydrogels, implants or nanofibers, have been developed to improve the adhesion, proliferation or differentiation of stem cells and to culture differentiated cells. This review article will help to inform the optimal choice of ECM protein-derived peptides for the development of scaffolds, implants, hydrogels, nanofibers and 2D cell culture dishes to regulate the proliferation and direct the differentiation of stem cells into specific lineages.
Asunto(s)
Proteínas de la Matriz Extracelular , Péptidos , Humanos , Péptidos/química , Diferenciación Celular , Integrinas/metabolismo , Células Madre/metabolismo , Proliferación Celular , HidrogelesRESUMEN
Human pluripotent stem cells (hPSCs) have the ability to differentiate into cells derived from three germ layers and are an attractive cell source for cell therapy in regenerative medicine. However, hPSCs cannot be cultured on conventional tissue culture flasks but can be cultured on biomaterials with specific hPSC integrin interaction sites. We designed hydrogels conjugated with several designed peptides that had laminin-ß4 active sites, optimal elasticities and different zeta potentials. A higher expansion fold of hPSCs cultured on the hydrogels was found with the increasing zeta potential of the hydrogels conjugated with designed peptides, where positive amino acid (lysine) insertion into the peptides promoted higher zeta potentials of the hydrogels and higher expansion folds of hPSCs when cultured on the hydrogels using xeno-free protocols. The hPSCs cultured on hydrogels conjugated with the optimal peptides showed a higher expansion fold than those on recombinant vitronectin-coated plates, which are the gold standard of hPSC cultivation dishes. The hPSCs could differentiate into specific cell lineages, such as mesenchymal stem cells (MSCs) and MSC-derived osteoblasts, even after being cultivated on hydrogels conjugated with optimal peptides for long periods of time, such as 10 passages.
Asunto(s)
Hidrogeles , Células Madre Pluripotentes , Humanos , Hidrogeles/química , Proliferación Celular , Células Madre Pluripotentes/metabolismo , Péptidos/farmacología , Péptidos/metabolismo , Diferenciación CelularRESUMEN
Human pluripotent stem cells (hPSCs) can be proliferated on completely synthetic materials under xeno-free cultivation conditions using biomaterials grafted with extracellular matrix protein (ECM)-derived peptides. However, cell culture biomaterials grafted with ECM-derived peptides must be prepared using a high concentration of peptide reaction solution (e.g. 1000 µg/ml), whereas the ECM concentration of the ECM-coated surface for hPSC culture is typically 5 µg/ml. We designed a polyethylene glycol (PEG) joint nanosegment (linker) to be used between base cell culture biomaterials and bioactive ECM-derived peptides to enhance the probability of contact between ECM-derived peptides and cell binding receptors of hPSCs. Vitronectin-derived peptides with glycine joint nanosegments (GCGG) were conjugated onto poly (vinyl alcohol-co-itaconic acid) hydrogels via PEG joint nanosegments, and human embryonic stem cells (hESCs) were cultivated on these hydrogels. hESCs could successfully be cultivated on hydrogels while maintaining their pluripotency and differentiation potential to differentiate into cells that are induced from three germ layers in vitro and in vivo, where only a 50 µg/ml ECM-derived peptide concentration was used when the PEG joint nanosegments were introduced into peptides that were grafted onto hydrogel surfaces. The joint nanosegments between bioactive peptides and base cell culture biomaterials were found to contribute to efficient hESC attachment and proliferation.
Asunto(s)
Células Madre Embrionarias Humanas , Hidrogeles , Humanos , Polietilenglicoles , Proteínas de la Matriz Extracelular , Péptidos/farmacología , Alcohol Polivinílico , Materiales Biocompatibles/farmacología , Células CultivadasRESUMEN
Stem cells serve as an ideal source of tissue regeneration therapy because of their high stemness properties and regenerative activities. Mesenchymal stem cells (MSCs) are considered an excellent source of stem cell therapy because MSCs can be easily obtained without ethical concern and can differentiate into most types of cells in the human body. We prepared cell culture materials combined with synthetic polymeric materials of poly-N-isopropylacrylamide-co-butyl acrylate (PN) and extracellular matrix proteins to investigate the effect of cell culture biomaterials on the differentiation of dental pulp stem cells (DPSCs) into neuronal cells. The DPSCs cultured on poly-L-ornithine (PLO)-coated (TPS-PLO) plates and PLO and PN-coated (TPS-PLO-PN) plates showed excellent neuronal marker (ßIII-tubulin and nestin) expression and the highest expansion rate among the culture plates investigated in this study. This result suggests that the TPS-PLO and TPS-PN-PLO plates maintained stable DPSCs proliferation and had good capabilities of differentiating into neuronal cells. TPS-PLO and TPS-PN-PLO plates may have high potentials as cell culture biomaterials for the differentiation of MSCs into several neural cells, such as cells in the central nervous system, retinal cells, retinal organoids and oligodendrocytes, which will expand the sources of cells for stem cell therapies in the future.
RESUMEN
The transplantation of human mesenchymal stem cells (hMSCs), such as bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs), has shown beneficial effects in protecting transplanted tissues and cells against graft-versus-host disease (GVHD). Human pluripotent stem cell (hPSC)-derived mesenchymal stem cells (MSCs) can also be used to generate hMSCs with stable characteristics without limitations. Therefore, we differentiated human induced pluripotent stem cells (hiPSCs, H-M5) and human embryonic stem cells (hESCs, H9) into hMSCs on dishes coated with different extracellular matrix (ECM) proteins to study the effect of cell culture biomaterials on hPSC differentiation into hMSCs. hPSC-derived MSCs cultured on Matrigel (MAT)-coated, collagen (COL)-coated and laminin-521 (LN-521)-coated tissue culture polystyrene (TCP) dishes showed excellent proliferation speed and reduced aging over 10 passages. High MSC surface marker (CD44, CD73, CD90 and CD105) expression was also observed on hPSC-derived MSCs cultured on MAT-coated, COL-coated and LN-521-coated TCP dishes as well as uncoated TCP dishes. Analysis of late osteogenic differentiation by evaluation of mineral deposition revealed that hPSC-derived MSCs cultured on fibronectin (FN)-coated and LN-521-coated TCP dishes showed high osteogenic differentiation. ECM proteins are effective as coating materials on cell culture biomaterials to regulate the proliferation and differentiation fate of hPSC-derived MSCs.
Asunto(s)
Diferenciación Celular , Proteínas de la Matriz Extracelular , Células Madre Pluripotentes Inducidas , Células Madre Mesenquimatosas , Materiales Biocompatibles/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Mesenquimatosas/citología , OsteogénesisRESUMEN
[This corrects the article DOI: 10.1016/j.bioactmat.2020.08.028.].
RESUMEN
Human pluripotent stem cells (hPSCs) are typically cultivated on extracellular matrix (ECM) protein-coated dishes in xeno-free culture conditions. We supplemented mixed ECM proteins (laminin-511 and recombinant vitronectin, rVT) in culture medium for hPSC culture on conventional polystyrene dishes. Three hPSC cell lines were successfully cultivated on uncoated polystyrene dishes in medium supplemented with optimal conditions of laminin-511 and rVT. Excellent colony shape and colony size as well as high expansion fold of hPSCs were found under these conditions, whereas the colony size was small and poor expansion fold was found solely on L-511-coated dishes. A small portion of L-511 in the culture medium supported hPSC adhesion and prevented the adhesion from being too strong on the uncoated dishes, and rVT in the culture medium further supported adhesion of hPSCs on the dishes by maintaining their pluripotency. Having the optimal composition of L-511 and rVT in the culture medium was important for generating good hPSC colony shapes and sizes as well as a high expansion fold. After long-term culture of hPSCs on uncoated dishes supplemented with the mixed proteins, the hPSCs successfully showed pluripotent markers and could differentiate into a specific lineage of cells, cardiomyocytes, with high efficiency.
Asunto(s)
Laminina/metabolismo , Células Madre Pluripotentes/metabolismo , Poliestirenos/química , Vitronectina/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Humanos , Tamaño de la Partícula , Proteínas Recombinantes/metabolismo , Propiedades de SuperficieRESUMEN
Cancer stem cells (CSCs) or cancer-initiating cells (CICs) are key factors for tumor generation and metastasis. We investigated a filtration method to enhance CSCs (CICs) from colon carcinoma HT-29 cells and primary colon carcinoma cells derived from patient colon tumors using poly(lactide-co-glycolic acid)/silk screen (PLGA/SK) filters. The colon carcinoma cell solutions were permeated via porous filters to obtain a permeation solution. Then, the cell cultivation media were permeated via the filters to obtain the recovered solution, where the colon carcinoma cells that adhered to the filters were washed off into the recovered solution. Subsequently, the filters were incubated in the culture media to obtain the migrated cells via the filters. Colon carcinoma HT-29 cells with high tumorigenicity, which might be CSCs (CICs), were enhanced in the cells in the recovered solution and in the migrated cells based on the CSC (CIC) marker expression, colony-forming unit assay, and carcinoembryonic antigen (CEA) production. Although primary colon carcinoma cells isolated from colon tumor tissues contained fibroblast-like cells, the primary colon carcinoma cells were purified from fibroblast-like cells by filtration through PLGA/SK filters, indicating that the filtration method is effective in purifying primary colon carcinoma cells.
RESUMEN
We developed poly(vinyl alcohol-co-itaconic acid) (PV) hydrogels grafted with laminin-derived peptides that had different joint segments and several specific designs, including dual chain motifs. PV hydrogels grafted with a peptide derived from laminin-ß4 (PMQKMRGDVFSP) containing a joint segment, dual chain motif and cationic amino acid insertion could attach human pluripotent stem (hPS) cells and promoted high expansion folds in long-term culture (over 10 passages) with low differentiation rates, whereas hPS cells attached poorly on PV hydrogels grafted with laminin-α5 peptides that had joint segments with and without a cationic amino acid or on PV hydrogels grafted with laminin-ß4 peptides containing the joint segment only. The inclusion of a cationic amino acid in the laminin-ß4 peptide was critical for hPS cell attachment on PV hydrogels, which contributed to the zeta potential shifting to higher values (3-4 mV enhancement). The novel peptide segment-grafted PV hydrogels developed in this study supported hPS cell proliferation, which induced better hPS cell expansion than recombinant vitronectin-coated dishes (gold standard of hPS cell culture dishes) in xeno-free culture conditions. After long-term culture on peptide-grafted hydrogels, hPS cells could be induced to differentiate into specific lineages of cells, such as cardiomyocytes, with high efficiency.