Capturing totipotency in human cells through spliceosomal repression.

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.

Glioblastoma Stem Cell Targeting Peptide Isolated Through Phage Display Binds Cadherin 2.

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.

Modeling the Growth and Size Distribution of Human Pluripotent Stem Cell Clusters in Culture.

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.

[Experimental evaluation of the efficacy of tissue-engineered constructs in the treatment of limbal stem cell deficiency]. / Eksperimental'naya otsenka effektivnosti primeneniya tkaneinzhenernoi konstruktsii v lechenii limbal'noi nedostatochnosti.

Limbal stem cell deficiency (LSCD) is one of the leading factors negatively affecting the success of keratoplasty, and its treatment remains an urgent problem in ophthalmology. With the development of regenerative medicine, one of the promising approaches is the transplantation of tissue-engineered constructs from cultured limbal stem cells (LSCs) in biopolymer carriers. PURPOSE: This study was conducted to develop an experimental model of LSCD and evaluate the effectiveness of transplantation of a tissue-engineered construct consisting of cultured cells containing a population of LSCs and a collagen carrier. MATERIAL AND METHODS: The study was performed on 12 rabbits and included several stages. At the first stage, the physiological effects of collagen matrix implantation into the limbal zone were studied. At the second stage, tissue-engineered constructs consisting of LSCs on a collagen matrix were formed and their effect on the regeneration processes in the experimental LSCD model was analyzed. The animals were divided into 2 groups: surgical treatment (transplantation of the tissue-engineered construct) was used in the experimental group, and conservative treatment was used in the control group. Slit-lamp biomicroscopy with photo-registration, fluorescein corneal staining, optical coherence tomography of the anterior segment of the eye, and impression cytology were used to assess the results. RESULTS: No side reactions were observed after implantation of the collagen matrix into the limbal zone. One month after surgical treatment of the LSCD model in the experimental group, complete epithelization with minor manifestations of epitheliopathy was observed. In the control group, erosion of the corneal epithelium was noted. The time of corneal epithelization in the experimental and control groups was 9.2±2.95 and 46.20±12.07 days, respectively (p=0.139). According to the data of impression cytology, in the experimental group there were no goblet cells in the central part of the cornea, which indicates the restoration of corneal type epithelial cells, in contrast to the control group. CONCLUSION: Transplantation of a tissue-engineered construct from cultured limbal cells on a collagen membrane should be considered as a promising method for the treatment of limbal stem cell deficiency.

Cambial meristematic cell culture: a sustainable technology toward in vitro specialized metabolites production.

Cambial meristematic cells (CMCs) culture has received a fair share of scientific and industrial attention among the trending topics of plant cell culture, especially their potential toward secondary metabolites production. However, the conventional plant cell culture is often not commercially feasible because of difficulties associated with culture dedifferentiated cells. Several reports have been published to culture CMCs and bypass the dedifferentiation process in plant cell culture. Numerous mitochondria, multiple vacuoles, genetic stability, self-renewal, higher biomass, and stable metabolites accumulation are the characteristics features of CMCs compared with dedifferentiated cells (DDCs) culture. The CMCs culture has a broader application to produce large-scale natural compounds for: pharmaceuticals, food, and cosmetic industries. Cutting-edge progress in plant cellular and molecular biology has allowed unprecedented insights into cambial stem cell culture and its fundamental processes. Therefore, regarding sustainability and natural compound production, cambial cell culture ranks among the most vital biotechnological interventions for industrial and economic perspectives. This review highlights the recent advances in plant stem cell culture and understands the cambial cells induction and culture mechanisms that affect the growth and natural compounds production.

Male fertility preservation and restoration strategies for patients undergoing gonadotoxic therapies†.

Medical treatments for cancers or other conditions can lead to permanent infertility. Infertility is an insidious disease that impacts not only the ability to have a biological child but also the emotional well-being of the infertile individuals, relationships, finances, and overall health. Therefore, all patients should be educated about the effects of their medical treatments on future fertility and about fertility preservation options. The standard fertility preservation option for adolescent and adult men is sperm cryopreservation. Sperms can be frozen and stored for a long period, thawed at a later date, and used to achieve pregnancy with existing assisted reproductive technologies. However, sperm cryopreservation is not applicable for prepubertal patients who do not yet produce sperm. The only fertility preservation option available to prepubertal boys is testicular tissue cryopreservation. Next-generation technologies are being developed to mature those testicular cells or tissues to produce fertilization-competent sperms. When sperm and testicular tissues are not available for fertility preservation, inducing pluripotent stem cells derived from somatic cells, such as blood or skin, may provide an alternative path to produce sperms through a process call in vitro gametogenesis. This review describes standard and experimental options to preserve male fertility as well as the experimental options to produce functional spermatids or sperms from immature cryopreserved testicular tissues or somatic cells.

Directed differentiation and direct reprogramming: Applying stem cell technologies to hearing research.

Hearing loss is the most widely spread sensory disorder in our society. In the majority of cases, it is caused by the loss or malfunctioning of cells in the cochlea: the mechanosensory hair cells, which act as primary sound receptors, and the connecting auditory neurons of the spiral ganglion, which relay the signal to upper brain centers. In contrast to other vertebrates, where damage to the hearing organ can be repaired through the activity of resident cells, acting as tissue progenitors, in mammals, sensory cell damage or loss is irreversible. The understanding of gene and cellular functions, through analysis of different animal models, has helped to identify causes of disease and possible targets for hearing restoration. Translation of these findings to novel therapeutics is, however, hindered by the lack of cellular assays, based on human sensory cells, to evaluate the conservation of molecular pathways across species and the efficacy of novel therapeutic strategies. In the last decade, stem cell technologies enabled to generate human sensory cell types in vitro, providing novel tools to study human inner ear biology, model disease, and validate therapeutics. This review focuses specifically on two technologies: directed differentiation of pluripotent stem cells and direct reprogramming of somatic cell types to sensory hair cells and neurons. Recent development in the field are discussed as well as how these tools could be implemented to become routinely adopted experimental models for hearing research.

Tunable hydrogels for mesenchymal stem cell delivery: Integrin-induced transcriptome alterations and hydrogel optimization for human wound healing.

Therapeutic applications for mesenchymal stem/stromal cells (MSCs) are growing; however, the successful implementation of these therapies requires the development of appropriate MSC delivery systems. Hydrogels are ideally suited to cultivate MSCs but tuning hydrogel properties to match their specific in vivo applications remains a challenge. Thus, further characterization of how hydrogel-based delivery vehicles broadly influence MSC function and fate will help lead to the next generation of more intelligently designed delivery vehicles. To date, few attempts have been made to comprehensively characterize hydrogel impact on the MSC transcriptome. Herein, we have synthesized cell-degradable hydrogels based on bio-inert poly(ethylene glycol) tethered with specific integrin-binding small molecules and have characterized their resulting effect on the MSC transcriptome when compared with 2D cultured and untethered 3D hydrogel cultured MSCs. The 3D culture systems resulted in alterations in the MSC transcriptome, as is evident by the differential expression of genes related to extracellular matrix production, glycosylation, metabolism, signal transduction, gene epigenetic regulation, and development. For example, genes important for osteogenic differentiation were upregulated in 3D hydrogel cultures, and the expression of these genes could be partially suppressed by tethering an integrin-binding RGD peptide within the hydrogel. Highlighting the utility of tunable hydrogels, when applied to ex vivo human wounds the RGD-tethered hydrogel was able to support wound re-epithelialization, possibly due to its ability to increase PDGF expression and decrease IL-6 expression. These results will aid in future hydrogel design for a broad range of applications.

Transforming Growth Factor ß-Activated Kinase 1 Regulates Mesenchymal Stem Cell Proliferation Through Stabilization of Yap1/Taz Proteins.

Bone marrow-derived mesenchymal stem cells (BMMSCs) are multipotent stem cells capable of differentiation into a variety of cell types, proliferation, and production of clinically useful secretory factors. These advantages make BMMSCs highly useful for cell transplantation therapy. However, the molecular network underlying BMMSC proliferation remains poorly understood. Here, we showed that TGFß-activated kinase 1 (Tak1) is a critical molecule that regulates the activation of cell cycling and that Tak1 inhibition leads to quiescence in BMMSCs both in vivo and in vitro. Mechanistically, Tak1 was phosphorylated by growth factor stimulations, allowing it to bind and stabilize Yap1/Taz, which could then be localized to the nucleus. We also demonstrated that the quiescence induction by inhibiting Tak1 increased oxidized stress tolerance and improved BMMSC engraftment in intramuscular and intrabone marrow cell transplantation models. This study reveals a novel pathway controlling BMMSC proliferation and suggests a useful method to improve the therapeutic effect of BMMSC transplantation. Stem Cells 2019;37:1595-1605.

Gene Expression Regulation and Secretory Activity of Mesenchymal Stem Cells upon In Vitro Contact with Microarc Calcium Phosphate Coating.

The manufacture of biomaterial surfaces with desired physical and chemical properties that can directly induce osteogenic differentiation without the need for biochemical additives is an excellent strategy for controlling the behavior of mesenchymal stem cells (MSCs) in vivo. We studied the cellular and molecular reactions of MSCs to samples with a double-sided calcium phosphate (CaP) coating and an average roughness index (Ra) of 2.4-4.6 µm. The study aimed to evaluate the effect of a three-dimensional matrix on the relative mRNA expression levels of genes associated with the differentiation and maturation of MSCs toward osteogenesis (RUNX2, BMP2, BMP6, BGLAP, and ALPL) under conditions of distant interaction in vitro. Correlations were revealed between the mRNA expression of some osteogenic and cytokine/chemokine genes and the secretion of cytokines and chemokines that may potentiate the differentiation of cells into osteoblasts, which indicates the formation of humoral components of the extracellular matrix and the creation of conditions supporting the establishment of hematopoietic niches.

Concise Review: Current Status of Three-Dimensional Organoids as Preclinical Models.

Three-dimensional (3D) cultures use the property of some cells to self-organize in matrices and generate structures that can be programmed to represent an organ or a pathology. Organoid cultures are the 3D cultivation of source tissue (ranging from cells to tissue fragments) in a support matrix and specialized media that nearly resembles the physiological environment. Depending on the source tissue, growth factors, and inhibitors provided, organoids can be programmed to recapitulate the biology of a system and progression of pathology. Organoids are genetically stable, and genetically amenable, making them very suitable tools to study tissue homeostasis and cancer. In this Review, we focus on providing recent technical advances from published literature to efficiently use organoids as a tool for disease modeling and therapeutics. Also, we discuss stem cell biology principles used to generate multiple organoids and their characteristics, with a brief description of methodology. A major theme of this review is to expand organoid applications to the study disease progression and drug response in different cancers. We also discuss shortcomings, limitations, and advantages of developed 3D cultures, with the rationale behind the methodology. Stem Cells 2018;36:1329-1340.

Isolation of Human Photoreceptor Precursors via a Cell Surface Marker Panel from Stem Cell-Derived Retinal Organoids and Fetal Retinae.

Loss of photoreceptor cells due to retinal degeneration is one of the main causes of blindness in the developed world. Although there is currently no effective treatment, cell replacement therapy using stem-cell-derived photoreceptor cells may be a feasible future treatment option. In order to ensure safety and efficacy of this approach, robust cell isolation and purification protocols must be developed. To this end, we previously developed a biomarker panel for the isolation of mouse photoreceptor precursors from the developing mouse retina and mouse embryonic stem cell cultures. In the current study we applied this approach to the human pluripotent stem cell (hPSC) system, and identified novel biomarker combinations that can be leveraged for the isolation of human photoreceptors. Human retinal samples and hPSC-derived retinal organoid cultures were screened against 242 human monoclonal antibodies using a high through-put flow cytometry approach. We identified 46 biomarkers with significant expression levels in the human retina and hPSC differentiation cultures. Human retinal cell samples, either from fetal tissue or derived from embryonic and induced pluripotent stem cell cultures, were fluorescence-activated cell sorted (FACS) using selected candidate biomarkers that showed expression in discrete cell populations. Enrichment for photoreceptors and exclusion of mitotically active cells was demonstrated by immunocytochemical analysis with photoreceptor-specific antibodies and Ki-67. We established a biomarker combination, which enables the robust purification of viable human photoreceptors from both human retinae and hPSC-derived organoid cultures. Stem Cells 2018;36:709-722.

Isolation, culture and analysis of adult subependymal neural stem cells.

Individual cells dissected from the subependymal neurogenic niche of the adult mouse brain proliferate in medium containing basic fibroblast growth factor (bFGF) and/or epidermal growth factor (EGF) as mitogens, to produce multipotent clonal aggregates called neurospheres. These cultures constitute a powerful tool for the study of neural stem cells (NSCs) provided that they allow the analysis of their features and potential capacity in a controlled environment that can be modulated and monitored more accurately than in vivo. Clonogenic and population analyses under mitogen addition or withdrawal allow the quantification of the self-renewing and multilineage potency of these cells and the identification of the mechanisms involved in these properties. Here, we describe a set of procedures developed and/or modified by our group including several experimental options that can be used either independently or in combination for the ex vivo assessment of cell properties of NSCs obtained from the adult subependymal niche.

Adult stem cells in the small intestine are intrinsically programmed with their location-specific function.

Differentiation and specialization of epithelial cells in the small intestine are regulated in two ways. First, there is differentiation along the crypt-villus axis of the intestinal stem cells into absorptive enterocytes, Paneth, goblet, tuft, enteroendocrine, or M cells, which is mainly regulated by WNT. Second, there is specialization along the cephalocaudal axis with different absorptive and digestive functions in duodenum, jejunum, and ileum that is controlled by several transcription factors such as GATA4. However, so far it is unknown whether location-specific functional properties are intrinsically programmed within stem cells or if continuous signaling from mesenchymal cells is necessary to maintain the location-specific identity of the small intestine. Using the pure epithelial organoid technique, we show that region-specific gene expression profiles are conserved throughout long-term cultures of both mouse and human intestinal stem cells and correlated with differential Gata4 expression. Furthermore, the human organoid culture system demonstrates that Gata4-regulated gene expression is only allowed in absence of WNT signaling. These data show that location-specific function is intrinsically programmed in the adult stem cells of the small intestine and that their differentiation fate is independent of location-specific extracellular signals. In light of the potential future clinical application of small intestine-derived organoids, our data imply that it is important to generate GATA4-positive and GATA4-negative cultures to regenerate all essential functions of the small intestine.

Primary human mesenchymal stem cell culture under xeno-free conditions using surface-modified cellulose nanofiber scaffolds.

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.

Bionano Interactions of Organosilica Nanoparticles with Myeloid Derived Immune Cells.

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.

Whitening and moisturizing enhancing effects of three-dimensional human adipose-derived mesenchymal stem cell-conditioned medium-containing cream.

BACKGROUND: High-functional cosmetic products combined with the concept of "treatment" cosmetics are being introduced to the market. Cosmetic products containing a skin-derived microbiome, a three-dimensional (3D) stem cell culture medium, and low-molecular-weight collagen are being introduced, and these products are leading the cosmeceutical market. We aimed to confirm the potential of a 3D stem cell culture medium-containing cream as a skin-whitening and moisturizing product. AIM: To determine the enhancing effects of a cream containing 3D adipose tissue-derived mesenchymal stem cell-conditioned media (3D ADMSC-CM) on whitening and moisturization. METHODS: The inhibitory activities of tyrosinase (TYR) and melanin were confirmed using 3D ADMSC-CM. Furthermore, hyaluronic acid expression in 3D ADMSC-CM was verified. The clinical efficacy of the cream containing 3D ADMSC-CM was established by evaluating its antioxidant properties and effects on skin tone, radiance, freckles, and moisturization. RESULTS: The use of 3D ADMSC-CM suppressed the inhibitory effects of TYR and melanin by approximately 24% and 33%, respectively, and increased the expression of hyaluronic acid synthase. A significant difference was observed after 4 weeks of using 3D ADMSC-CM in the skin antioxidant evaluation. After 2 and 4 weeks of use, skin tone and radiance increased and skin freckles decreased significantly. Under extremely cold and dry weather conditions, the use of the cream increased skin moisturization. CONCLUSIONS: The 3D ADMSC-CM cream evaluated in an environment similar to the human body was found to enhance skin whitening and moisturization and can therefore be used in the skin care and cosmetic industries as a biocosmetic product.

Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture System.

Purpose: 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). Patients and Methods: 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. Results: 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. Conclusion: Dynamic 3D culture is desirable for the large-scale expansion of undifferentiated human IPSCs.

Therapeutic strategies of three-dimensional stem cell spheroids and organoids for tissue repair and regeneration.

Three-dimensional (3D) stem cell culture systems have attracted considerable attention as a way to better mimic the complex interactions between individual cells and the extracellular matrix (ECM) that occur in vivo. Moreover, 3D cell culture systems have unique properties that help guide specific functions, growth, and processes of stem cells (e.g., embryogenesis, morphogenesis, and organogenesis). Thus, 3D stem cell culture systems that mimic in vivo environments enable basic research about various tissues and organs. In this review, we focus on the advanced therapeutic applications of stem cell-based 3D culture systems generated using different engineering techniques. Specifically, we summarize the historical advancements of 3D cell culture systems and discuss the therapeutic applications of stem cell-based spheroids and organoids, including engineering techniques for tissue repair and regeneration.

Biomimetic Culture Strategies for the Clinical Expansion of Mesenchymal Stromal Cells.

Mesenchymal stromal/stem cells (MSCs) typically require significant ex vivo expansion to achieve the high cell numbers required for research and clinical applications. However, conventional MSC culture on planar (2D) plastic surfaces has been shown to induce MSC senescence and decrease cell functionality over long-term proliferation, and usually, it has a high labor requirement, a high usage of reagents, and therefore, a high cost. In this Review, we describe current MSC-based therapeutic strategies and outline the important factors that need to be considered when developing next-generation cell expansion platforms. To retain the functional value of expanded MSCs, ex vivo culture systems should ideally recapitulate the components of the native stem cell microenvironment, which include soluble cues, resident cells, and the extracellular matrix substrate. We review the interplay between these stem cell niche components and their biological roles in governing MSC phenotype and functionality. We discuss current biomimetic strategies of incorporating biochemical and biophysical cues in MSC culture platforms to grow clinically relevant cell numbers while preserving cell potency and stemness. This Review summarizes the current state of MSC expansion technologies and the challenges that still need to be overcome for MSC clinical applications to be feasible and sustainable.