Stem cell culture often requires various animal-derived components such as serum and collagen. This limits its practical use. Therefore, xeno-free (xenogeneic component-free) culture systems are receiving increased attention. Herein, we propose xeno-free, plant-derived cellulose nanofibers (CNFs) with different surface chemistry: 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized CNFs (TOCNFs) with carboxy groups and surface-sulfated CNFs (S-CNFs) for the proliferation of human mesenchymal stem cells (hMSCs) under various serum conditions. We cultured bone marrow-derived hMSCs on CNF scaffolds with various fiber lengths and functional group contents. Original CNFs were bioinert materials that did not contribute to cell adhesion. In contrast, the surface-modified CNFs facilitated the proliferation of immortalized hMSCs under normal and low-serum conditions. The TOCNFs (COONa: 1.47 mmol g-1; length: 0.53 μm), the S-CNFs (OSO3Na: 0.64 mmol g-1; 0.61 μm), and a combination of the two (1:1 by weight) enabled immortalized hMSCs to maintain their multipotency, even under serum-free conditions. Primary cultured hMSCs proliferated well on the TOCNF/S-CNF scaffolds in a completely serum-free medium, comparable to animal-derived type I collagen, although few hMSCs adhered to the standard polystyrene substrate. Our strategy of using surface-modified CNFs will inform the development of xeno-free culture systems to avoid the use of animal-derived materials for both cell culture media and scaffolds.

Investigating the interactions between nanomaterials and the cells they are likely to encounter in vivo is a critical aspect of designing nanomedicines for imaging and therapeutic applications. Immune cells such as dendritic cells, macrophages, and myeloid derived suppressor cells have a frontline role in the identification and removal of foreign materials from the body, with interactions shown to be heavily dependent on variables such as nanoparticle size, charge, and surface chemistry. Interactions such as cellular association or uptake of nanoparticles can lead to diminished functionality or rapid clearance from the body, making it critical to consider these interactions when designing and synthesizing nanomaterials for biomedical applications ranging from drug delivery to imaging and biosensing. We investigated the interactions between PEGylated organosilica nanoparticles and naturally endocytic immune cells grown from stem cells in murine bone marrow. Specifically, we varied the particle size from 60 nm up to 1000 nm and investigated the effects of size on immune cell association, activation, and maturation with these critical gatekeeper cells. These results will help inform future design parameters for in vitro and in vivo biomedical applications utilizing organosilica nanoparticles.

Human pluripotent stem cells (hPSCs) hold promise for regenerative medicine to replace essential cells that die or become dysfunctional. In some cases, these cells can be used to form clusters whose size distribution affects the growth dynamics. We develop models to predict cluster size distributions of hPSCs based on several plausible hypotheses, including (0) exponential growth, (1) surface growth, (2) Logistic growth, and (3) Gompertz growth. We use experimental data to investigate these models. A partial differential equation for the dynamics of the cluster size distribution is used to fit parameters (rates of growth, mortality, etc.). A comparison of the models using their mean squared error and the Akaike Information criterion suggests that Models 1 (surface growth) or 2 (Logistic growth) best describe the data.

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

UNASSIGNED: Two-dimensional (2D)-based cell culture systems, limited by their inherent heterogeneity and scalability, are a bottleneck in the production of high-quality cells for downstream biomedical applications. Finding the optimal conditions for large-scale stem cell culture while maintaining good cellular status is challenging. The aim of this study was to assess the effects of three-dimensional (3D) culture on the viability, proliferation, self-renewal, and differentiation of human induced pluripotent stem cells (IPSCs).
UNASSIGNED: Various culture conditions were evaluated to determine the optimal conditions to maintain the viability and proliferation of human IPSCs in a 3D environment: static versus dynamic culture, type of adhesion protein added to alginate (Matrigel™ versus gelatin), and the addition of Y-27632t on long-term 3D culture. The proliferation ability of the cells was evaluated via the MTS proliferation assay; the expression levels of the pluripotency markers Nanog and Oct3/4, PAX6 as an ectoderm marker, and laminin-5 and fibronectin as markers of extracellular matrix synthesis were assessed; and HIF1α and HIF2α levels were measured using quantitative reverse transcription polymerase chain reaction.
UNASSIGNED: Using a high-aspect-ratio vessel bioreactor with a gentle, low-sheer, and low-turbulence environment with sufficient oxygenation and effective mass transfer of nutrients and waste, we verified its ability to promote cell proliferation and self-renewal. The findings showed that human IPSCs have the ability to maintain pluripotency in a feeder-free system and by inhibiting ROCK signaling and using hypoxia to improve single-cell viability in 3D culture. Furthermore, these cells demonstrated increased self-renewal and proliferation when inoculated as single cells in 3D alginate beads by adding RI during the culture period.
UNASSIGNED: Dynamic 3D culture is desirable for the large-scale expansion of undifferentiated human IPSCs.

Application of autologous adipose-derived stem cells (ASC) for diabetic chronic wounds has become an emerging treatment option. However, ASCs from diabetic individuals showed impaired cell function and suboptimal wound healing effects. We proposed that adopting a low-glucose level in the culture medium for diabetic ASCs may restore their pro-healing capabilities.
ASCs from diabetic humans and mice were retrieved and cultured in high-glucose (HG, 4.5 g/L) or low-glucose (LG, 1.0 g/L) conditions. Cell characteristics and functions were investigated in vitro. Moreover, we applied diabetic murine ASCs cultured in HG or LG condition to a wound healing model in diabetic mice to compare their healing capabilities in vivo.
Human ASCs exhibited decreased cell proliferation and migration with enhanced senescence when cultured in HG condition in vitro. Similar findings were noted in ASCs derived from diabetic mice. The inferior cellular functions could be partially recovered when they were cultured in LG condition. In the animal study, wounds healed faster when treated with HG- or LG-cultured diabetic ASCs relative to the control group. Moreover, higher collagen density, more angiogenesis and cellular retention of applied ASCs were found in wound tissues treated with diabetic ASCs cultured in LG condition.
In line with the literature, our study showed that a diabetic milieu exerts an adverse effect on ASCs. Adopting LG culture condition is a simple and effective approach to enhance the wound healing capabilities of diabetic ASCs, which is valuable for the clinical application of autologous ASCs from diabetic patients.

Delivery of growth factors (GFs) is challenging for regulation of cell proliferation and differentiation due to their rapid inactivation under physiological conditions. Here, a bioactive polyelectrolyte multilayer (PEM) is engineered by the combination of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and glycosaminoglycans to be used as reservoir for GF storage. PNIPAM-grafted-chitosan (PChi) with two degrees of substitution (DS) are synthesized, namely LMW* (DS 0.14) and HMW (DS 0.03), by grafting low (2 kDa) and high (10 kDa) molecular weight of PNIPAM on the backbone of chitosan (Chi) to be employed as polycations to form PEM with the polyanion heparin (Hep) at pH 4. Subsequently, PEMs are chemically crosslinked to improve their stability at physiological pH 7.4. Resulting surface and mechanical properties indicate that PEM containing HMW is responsive to temperature at 20 °C and 37 °C, while LMW is not. More importantly, Hep as terminal layer combined with HMW allows not only a better retention of the adhesive protein vitronectin but also a sustained release of FGF-2 at 37 °C. With the synergistic effect of vitronectin and matrix-bound FGF-2, significant promotion on adhesion, proliferation, and migration of 3T3 mouse embryonic fibroblasts is achieved on HMW-containing PEM compared to Chi-containing PEM and exogenously added FGF-2. Thus, PEM containing PNIPAM in combination with bioactive glycosaminoglycans like Hep represents a versatile approach to fabricate a GF delivery system for efficient cell culture, which can be potentially served as cell culture substrate for production of (stem) cells and bioactive wound dressing for tissue regeneration.

Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of precultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy.

Glioblastoma stem cells (GSCs) have unique properties of self-renewal and tumor initiation that make them potential therapeutic targets. Development of effective therapeutic strategies against GSCs requires both specificity of targeting and intracranial penetration through the blood-brain barrier. We have previously demonstrated the use of in vitro and in vivo phage display biopanning strategies to isolate glioblastoma targeting peptides. Here we selected a 7-amino acid peptide, AWEFYFP, which was independently isolated in both the in vitro and in vivo screens and demonstrated that it was able to target GSCs over differentiated glioma cells and non-neoplastic brain cells. When conjugated to Cyanine 5.5 and intravenously injected into mice with intracranially xenografted glioblastoma, the peptide localized to the site of the tumor, demonstrating intracranial tumor targeting specificity. Immunoprecipitation of the peptide with GSC proteins revealed Cadherin 2 as the glioblastoma cell surface receptor targeted by the peptides. Peptide targeting of Cadherin 2 on GSCs was confirmed through ELISA and in vitro binding analysis. Interrogation of glioblastoma databases demonstrated that Cadherin 2 expression correlated with tumor grade and survival. These results confirm that phage display can be used to isolate unique tumor-targeting peptides specific for glioblastoma. Furthermore, analysis of these cell specific peptides can lead to the discovery of cell specific receptor targets that may serve as the focus of future theragnostic tumor-homing modalities for the development of precision strategies for the treatment and diagnosis of glioblastomas.

The capability of pluripotent embryonic stem cells (ESCs) to proliferate and differentiate into specific lineages makes them an important avenue of research in the field of cell therapy as well as a useful model to study patterns of differentiation and gene expression, recapitulating many events that occur during the very early stages of development of the mammalian embryo. With striking similarities that exist between inherently programmed embryonic development of the nervous system in vivo and the differentiation of ESCs in vitro, they have already been used to treat locomotive and cognitive deficits caused by brain injury in rodents. A suitable differentiation model thus empowers us with all these opportunities. In this chapter, we describe a neural differentiation model from mouse embryonic stem cells using retinoic acid as the inducer. This method is among the most commonly used one to acquire a homogeneous population of neuronal progenitor cells or mature neurons as desired. The method is scalable, efficient, and results in production of ~70% neural progenitor cells within 4-6 days.