3D Stem Cell Culture Research Articles

An Advocacy for the Use of 3D Stem Cell Culture Systems for the Development of Regenerative Medicine: An Emphasis on Photoreceptor Generation

The availability of stem cells is of great promise to study early developmental stages and to generate adequate cells for cell transfer therapies. Although many researchers using stem cells were successful in dissecting intrinsic and extrinsic mechanisms and in generating specific cell phenotypes, few of the stem cells or the differentiated cells show the capacity to repair a tissue. Advances in cell and stem cell cultivation during the last years made tremendous progress in the generation of bona fide differentiated cells able to integrate into a tissue after transplantation, opening new perspectives for developmental biology studies and for regenerative medicine. In this review, we focus on the main works attempting to create in vitro conditions mimicking the natural environment of CNS structures such as the neural tube and its development in different brain region areas including the optic cup. The use of protocols growing cells in 3D organoids is a key strategy to produce cells resembling endogenous ones. An emphasis on the generation of retina tissue and photoreceptor cells is provided to highlight the promising developments in this field. Other examples are presented and discussed, such as the formation of cortical tissue, the epithelial gut or the kidney organoids. The generation of differentiated tissues and well-defined cell phenotypes from embryonic stem (ES) cells or induced pluripotent cells (iPSCs) opens several new strategies in the field of biology and regenerative medicine. A 3D organ/tissue development in vitro derived from human cells brings a unique tool to study human cell biology and pathophysiology of an organ or a specific cell population. The perspective of tissue repair is discussed as well as the necessity of cell banking to accelerate the progress of this promising field.

3D culture of murine neural stem cells on decellularized mouse brain sections

Transplantation of neural stem cells (NSC) in diseased or injured brain tissue is widely studied as a potential treatment for various neurological pathologies. However, effective cell replacement therapy relies on the intrinsic capacity of cellular grafts to overcome hypoxic and/or immunological barriers after transplantation. In this context, it is hypothesized that structural support for grafted NSC will be of utmost importance. With this study, we present a novel decellularization protocol for 1.5 mm thick mouse brain sections, resulting in the generation of acellular three-dimensional (3D) brain sections. Next, the obtained 3D brain sections were seeded with murine NSC expressing both the eGFP and luciferase reporter proteins (NSC-eGFP/Luc). Using real-time bioluminescence imaging, the survival and growth of seeded NSC-eGFP/Luc cells was longitudinally monitored for 1–7 weeks in culture, indicating the ability of the acellular brain sections to support sustained ex vivo growth of NSC. Next, the organization of a 3D maze-like cellular structure was examined using confocal microscopy. Moreover, under mitogenic stimuli (EGF and hFGF-2), most cells in this 3D culture retained their NSC phenotype. Concluding, we here present a novel protocol for decellularization of mouse brain sections, which subsequently support long-term 3D culture of undifferentiated NSC.

3D mouse embryonic stem cell culture for generating inner ear organoids.

This protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 96-well plates in medium containing extracellular matrix proteins to promote epithelialization. During the first 14 d, a series of precisely timed protein and small-molecule treatments sequentially induce epithelia that represent the mouse embryonic non-neural ectoderm, preplacodal ectoderm and otic vesicle epithelia. Ultimately, these tissues develop into cysts with a pseudostratified epithelium containing inner ear hair cells and supporting cells after 16-20 d. Concurrently, sensory-like neurons generate synapse-like structures with the derived hair cells. We have designated the stem cell-derived epithelia harboring hair cells, supporting cells and sensory-like neurons as inner ear organoids. This method provides a reproducible and scalable means to generate inner ear sensory tissue in vitro.

3D Culture of rat embryonic stem cells and rat induced pluripotent stem cells in a novel PGmatrix hydrogel (LB20)

Expanding embryonic stem cells (ESCs) in a synthetic 3D microenvironment in vitro while maintaining the undifferentiated state has been challenging. It would be advantageous to identify a substrate which could be modified such as by changing ligands that interact with cell surface receptors or by changing the substrate’s stiffness. We developed a synthetic PGmatrix hydrogel to include these design specifications and determined whether it could support the expansion of undifferentiated ESCs in a 3D microenvironment. Rat ESCs (rESCs) and rat induced pluripotent stem cells (riPSCs) were used for evaluation. The cells were grown both in 2D and 3D in PGmatrix hydrogel containing 2 inhibitors. Dynamic rheological tests of PGmatrix hydrogel in 2i medium showed a rapid increase of storage moduli (G’ = 600 Pa for 3mM in 1 hr) indicating gelation after mixing. The mechanical strength of hydrogel was full recovered after shear‐thinning into liquid phase.Also, the hydrogel reached a self‐supporting strength within 1000sec which is a reasonable rate to ensure cells were suspended in the hydrogel matrix before they reached the bottom of the plate. Both cell lines were cultured for more than 30 passages using regular trypsinization method to produce single cells at passage prior to plating. We harvested cells from the hydrogel for passaging by shearing the gel to liberate the cells. Phase contrast images of cells grown in 3D showed similar morphology which had rounded phase‐bright borders compared to cells grown on murine embryonic fibroblast (MEF) feeder layer. They retained pluripotent stem cell properties and showed positive staining for alkaline phosphatase, Oct‐4, Nanog, Sox‐2 and SSEA‐1. Karyotypes of pluripotent stem cells cultured in 3D were normal after 20 passages. Embryoid bodies (EBs) could be formed by hanging drop after the cells were trypsinized from 3D cultures. We plan to compare the gene expression profiles of 2D and 3D cells and determine if the 3D cultured cells can undergo differentiation into all the 3 germ layers and form teratomas. Growing ESCs in a fully synthetic 3D culture can significant advantages for directing differentiation and examining the interactions with surface receptors. Such knowledge will enable 3D culture for their use in drug development or for encapsulation of cells for clinical use.

The miR-7 Identified from Collagen Biomaterial-Based Three-Dimensional Cultured Cells Regulates Neural Stem Cell Differentiation

Increasing evidence suggests that three-dimensional (3D) cultures provide more appropriate microenvironments to control stem cell response compared with traditional two-dimensional (2D) cultures. However, the molecular mechanism involved in 3D cultured stem cells is not well known. Several microRNAs whose target genes involved in the regulation of self-renewal and differentiation of stem cells were found to be downregulated in 3D cultured PA-1 cells. Among them, miR-7 was predicted to target Kruppel-like factor 4 (Klf4), a key gene for self-renewal of neural stem cells (NSCs). We showed that the differentiation of NSCs was inhibited in 3D collagen scaffolds compared with 2D cultured cells. The quantitative real-time PCR (qPCR) analysis indicated that the expression of miR-7 and Klf4 changed significantly in 2D cultures, whereas the expression stability of miR-7 and Klf4 was detected in 3D cultures. Using luciferase assay and western blot, Klf4 was identified as a target of miR-7 indicating that miR-7 plays a critical role in maintaining the self-renewal capacity through a Klf4-dependent mechanism in 3D cultured cells. Thus, the collagen scaffold-based 3D cell cultures may provide a platform to reveal the regulatory mechanism of cell regulators, which are difficult to find in traditional 2D cell cultures.

Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture

The inner ear contains sensory epithelia that detect head movements, gravity and sound. It is unclear how to derive these sensory epithelia from pluripotent stem cells, a process which will be critical for modeling inner ear disorders or developing cell-based therapies for profound hearing loss and balance disorders1,2. To date, attempts to derive inner ear mechanosensitive hair cells and sensory neurons have resulted in inefficient or incomplete phenotypic conversion of stem cells into inner ear-like cells3–7. A key insight lacking from these previous studies is the importance of the non-neural and pre-placodal ectoderm, two critical precursors during inner ear development8–11. Here we report the step-wise differentiation of inner ear sensory epithelia from mouse embryonic stem cells (ESCs) in three-dimensional culture12,13. We show that by recapitulating in vivo development with precise temporal control of BMP, TGFβ and FGF signaling, ESC aggregates transform sequentially into non-neural, pre-placodal and otic placode-like epithelia. Remarkably, in a self-organized process that mimics normal development, vesicles containing prosensory cells emerge from the presumptive otic placodes and give rise to hair cells bearing stereocilia bundles and a kinocilium. Moreover, these stem cell-derived hair cells exhibit functional properties of native mechanosensitive hair cells and form specialized synapses with sensory neurons that have also arisen from ESCs in the culture. Finally, we demonstrate how these vesicles are structurally and biochemically comparable to developing vestibular end organs. Our data thus establish a novel in vitro model of inner ear differentiation that can be used to gain deeper insight into inner ear development and disorder.

Abstract 5545: Optimal drug concentration screening and evaluation in cancer stem cells & 3D tumor stem cell cultures drug response assays in association with clinical efficacy for pancreatic cancer stem cell.

Abstract Three-dimensional (3D) culture of cancer cell lines has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo. Moreover, new drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumors and to decrease the use of experimental animals. The induction of chemotherapy or concomitant chemoradiotherapy has been used to increasingly to improve survival, and organ preservation. This approach encounters significant morbidity and mortality. Therefore reliable chemosensitivity assays are needed to accurately predict the response to chemotherapy and guide the selection and treatment of cancer patients. The purpose of this study is to examine and evaluate optimum drug candidates in vitro chemosensitivity on patient tumor tissues directly in culture and on their Cancer stem cell cultures. The tumor samples obtained after surgery or biopsy, were placed immediately in Celprogen Tumor Transportation Media and shipped at 4-8 OC for processing. Tissues were washed with 1X PBS solution and aseptically cut into 0.5mm sections and cultured in 6 well tissue culture plates with an insert pre-coated with ECM. All cancer cell types remain viable and maintain their native architecture for at least 14 days and incorporated DNA measured by adding EdU(5-ethynyl-2’-deoxyuridine) to the culture. The efficacy of various therapeutic agents targeting major pathways (wnt,Notch,PI3K,MAPK,STAT) and chemotherapy agents were tested using DNA uptake and TUNNEL assay anti-cancer agents was calculated according to the inhibition index. The same compounds were tested for utilizing the patient's Pancreatic Cancer Stem Cell Cultures established with Celprogen's Media and ECM. Expression of PDX-1, SHH, CD24, CD44, CD133, EpCAM, CBX7, OCT4, SNAIL, SLUG, TWIST, Ki-67, E-cadherin, β-catenin and vimentin were quantified by qPCR or immunocytochemistry per cell culture. The epithelial-mesenchymal transition (EMT) is linked to induction of a stem-cell like phenotype. We cultured the cells in low oxygen since Tumor hypoxia induces EMT, which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Among the 600 compounds tested Gemcitabine, Taxol, Fluorouracil, Leucovorin, Irinotecan, and Oxaliptin were not effective against Pancreatic Cancer Stem cell (CSC) but were effective on tumor cells (differentiated CSCs). We were able to show 6 compounds that were effective against Pancreatic CSC targeting selected pathways. Citation Format: John P. Clery, Esteban Gomez, Michael Sharma, Aabha khemani, Cristian Sharma, Rubio Punzalan, miriam navel, Natalee Amezcua, Jitesh Jani, Jay Sharma. Optimal drug concentration screening and evaluation in cancer stem cells & 3D tumor stem cell cultures drug response assays in association with clinical efficacy for pancreatic cancer stem cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5545. doi:10.1158/1538-7445.AM2013-5545

Effects of Multiple Agents on Epithelial Differentiation of Rabbit Adipose-Derived Stem Cells in 3D Culture

Mesenchymal stem cells have been given particular attention in tissue regeneration research due to their multipotency and proliferative activity. In this study, we investigated the possibility of epithelial differentiation of rabbit adipose-derived stem cells (rASCs) in an in vitro 3D culture system. The experimental procedure was performed with different contributing factors including all-trans retinoic acid (ATRA), epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and hydrocortisone in air-liquid interface culture, for modulating proliferation and providing a synergistic effect on epithelial differentiation of rASCs. After induction, immunofluorescence staining, western blot analysis, flow cytometry analysis, and quantitative real-time polymerase chain reaction assay have been performed to detect the expression of epithelial-specific markers and mesenchymal marker alpha-smooth muscle actin (α-SMA). The growth pattern and viability of cells were evaluated by transmission electron microscopy and Hoechst 33258 assay, respectively. After treated with optimized induction medium (including 2.5 μM ATRA, 20 ng/mL EGF, 10 ng/mL KGF, 10 ng/mL HGF, and 0.5 μg/mL hydrocortisone), rASCs were observed to display a stratified epithelial-like morphology, with the expression of cytokeratin 19 and cytokeratin 13 in 63.69%±2.63% and 22.17%±1.51%, respectively, and the relative expression level of cytokeratin 19 increased to 3.152 compared with 0.151 before induction. The expression of α-SMA decreased to 19.40%±1.45% after induction, but almost no expression of involucrin was detected. The results showed that the establishment of an epithelial-specific microenvironment may be a feasible way for epithelial differentiation of ASCs in vitro, and provided an alternative for research on epithelium regeneration.

CARS and SHG microscopy to follow collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin hydrogel 3D cultures

Coherent anti‐Stokes Raman scattering (CARS) microscopy is used in conjunction with second harmonic generation (SHG) to follow the early stage of stem cell differentiation within a three‐dimensional (3D) scaffold. Formation of an extracellular matrix mainly composed of collagen is one of the first signs of human mesenchymal stem cell (hMSC) differentiation. This work shows that the multimodal CARS and SHG constitutes a powerful noninvasive, label‐free technique for monitoring collagen production in 3D cell cultures. Its simultaneous imaging of cell morphology and the distribution of the collagen produced by living cells during long‐term (4 weeks) experiment furnished important information about the cell‐scaffold interaction and the establishment of the extracellular matrix, while its very low collagen detection limit permitted mapping of even the small quantity produced in a few hours. This finding indicates that the multimodal CARS and SHG imaging can be proposed as a new way of monitoring the differentiation of stem cells by evaluating their production of collagen in both short and long‐term experiments. Further evidence is also provided of the efficacy of fibrin hydrogel as a scaffold autoinducing the differentiation stimulus in 3D human mesenchymal stem cell cultures. Copyright © 2012 John Wiley & Sons, Ltd.

A comparative study of aggrecan synthesis between natural articular chondrocytes and differentiated chondrocytes from adipose derived stem cells in 3D culture

Introduction:The main obstacle for tissue engineering is to find the most appropriate cell which is able to produce extracellular matrix (ECM) similar or better than natural chondrocytes in vitro. This study compared aggrecan synthesis's potential between differentiated chondrocytes (DCs) from adipose-derived stem cells (ADSCs) and natural articular chondrocytes (NCs) in 3D culture in vitro.Materials and Methods:Human ADSCs were isolated from sub-cutaneous adipose tissue and then the surface markers including CD 14, 45 CD105, CD90, CD44 were analyzed by flow cytometry. Also human articular chondrocytes were yielded of non-weight bearing area of Knee cartilage. Both types of the cells were encapsulated in alginate scaffolds and cultured in chondrogenic medium with and without TGFβ3 for 3 weeks. Then the extent of aggercan (AGC) production was evaluated by ELISA on days 14 and 21.Results:Our findings indicated that differentiated chondrocytes (DCs) with and without TGFβ3 synthesized more AGC than natural chondrocytes (NCs) on day 14. But DCs without TGFβ3 had higher production than other groups on day 21. Application of TGFβ3 resulted in an increase of amount of AGC in DCs on day 14 but a decrease on day 21 than same group.Conclusion:Since, aggrecan is an important chondrogenic marker, it was concluded that ADSCs can be possible reliable alternative cell source for cartilage tissue engineering in future.

Impact of 3D culture of mesenchymal stem cells to repair expanding AAAS

Impact of 3D culture of mesenchymal stem cells to repair expanding AAAS

Impact of DAG stimulation on mineral synthesis, mineral structure and osteogenic differentiation of human cord blood stem cells

It remains unexplored in what way osteogenic stimulation with dexamethasone, ascorbic acid and β-glycerol phosphate (DAG) influences the process of mineralization, the composition and structure of the assembled mineral. Therefore, we analyzed and characterized biomineralization in DAG-stimulated and unstimulated 3D human unrestricted somatic stem cell (USSC) cultures. Microspheres were analyzed by histological staining, scanning electron microscopy (SEM), semi-quantitative energy-dispersive X-ray spectroscopy (EDX), quantitative wavelength-dispersive X-ray spectroscopy (WDX), transmission electron microscopy (TEM), selected area electron diffraction (SAED) and Raman spectroscopy. Mineral material was detected by SEM and histological staining in both groups, and showed structural differences. DAG influenced the differentiation of USSCs and the formation, structure and composition of the assembled mineral. SEM showed that cells of the + DAG spheres exhibited morphological signs of osteoblast-like cells. EDX and WDX confirmed a Ca–P mineral in both groups. Overall, the mineral material found showed structural similarities to the mineral substance of bony material.

Two- and Three-Dimensional Culture of Keratinocyte Stem and Precursor Cells Derived from Primary Murine Epidermal Cultures

In the skin, multipotent keratinocyte stem cells (KSC) are localised in the hair follicle bulge region. Although, KSC can be cultivated and grown in two-dimensional (2D) culture they rapidly lose stem cell markers when isolated from their niche. Currently, there is no KSC culture method available which recapitulates an environment similar to the KSC niche in the hair follicle. Here we describe the successful establishment of an in vitro 3D stem cell culture model developed from clonally growing keratinocyte lines derived from neonatal mice using culture conditions previously established for human keratinocytes. After 20 passages, keratinocyte lines showed a stable ratio of holoclones (stem cells), meroclones (stem and precursor cells) and paraclones (differentiating cells), with approximately 29% holoclones, 54% meroclones and 17% paraclones, and were thus termed keratinocyte stem and precursor cell (KSPC) cultures. In high calcium medium, KSPC cultures grown at the air-liquid interphase differentiated and formed epidermal equivalents. Notably, and in contrast to primary keratinocytes, keratinocytes from KSPC cultures were able to aggregate and form spherical clusters in hanging drops, a characteristic hallmark shared with other stem cell types. Similar to the in vivo situation in the hair follicle bulge, KSPC aggregates also showed low proliferation, down-regulation of keratin 6, absence of keratin 1, and expression of the KSC markers keratin 15, Sox9, NFATc1 and Zfp145. KSPC aggregates therefore provide an optimal in vitro 3D environment for the further characterisation and study of normal and genetically modified KSPC.

Retaining Cell-Cell Contact Enables Preparation and Culture of Spheroids Composed of Pure Primary Cancer Cells From Colorectal Cancer

Primary culture of the cancer cells from patients’ tumors can provide crucial information of individual tumors, yet the technology has not been optimized until now. We developed an innovative culture method for primary colorectal cancer cells, based on the principle that cell–cell contact of cancer cells was maintained throughout the process. When tumor tissue was dissociated into cell clusters, in which cell–cell contact was retained, they rapidly formed spheroids that we termed cancer tissue-originated spheroids (CTOSs). CTOSs of colorectal cancer consisted of highly purified and viable cancer cells, and they were prepared with high efficiency. In immunodeficient mice, CTOSs formed xenograft tumors that retained the features of the parental tumors. Moreover, CTOSs were able to be cultured and expanded in vitro using a 3D culture system and stem cell culture medium. This method allowed evaluation of chemosensitivity and signal pathway activation in cancer cells from individual patients. Easy preparation and culture of pure primary cancer cells provides an innovative platform for studying cancer biology and developing personalized medicine.

Abstract 4371: Optimal drug concentration screening and evaluation in cancer stem cells & 3D tumor stem cell cultures drug response assays in association with clinical efficacy for melanoma cancer stem cell

Abstract Optimal drug concentration screening and evaluation in Cancer stem cells & 3D Tumor Stem Cell cultures Drug Response assays in association with Clinical efficacy for Melanoma Cancer Stem Cell. Three-dimensional (3D) culture of cancer cell lines has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo. Moreover, new drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumors and to decrease the use of experimental animals. The induction of chemotherapy or concomitant chemoradiotherapy has been used to increasingly to improve survival, and organ preservation. This approach encounters significant morbidity and mortality. Therefore reliable chemosensitivity assays are needed to accurately predict the response to chemotherapy and guide the selection and treatment of cancer patients. The purpose of this study is to examine and evaluate optimum drug candidates in vitro chemosensitivity on patient tumor tissues directly in culture and on their Cancer stem cell cultures. The tumor samples obtained after surgery or biopsy, were placed immediately in Celprogen Tumor Transportation Media and shipped at 4-8 OC for processing. Tissues were washed with 1X PBS solution and aseptically cut into 0.5mm sections and cultured in 6 well tissue culture plates with an insert pre-coated with ECM. All cancer cell types remain viable and maintain their native architecture for at least 14 days and incorporated DNA measured by adding EdU(5-ethynyl-2’-deoxyuridine) to the culture. The efficacy of various therapeutic agents targeting major pathways (wnt, Notch, PI3K, MAPK, STAT) and chemotherapy agents were tested using DNA uptake and TUNNEL assay anti-cancer agents was calculated according to the inhibition index. The same compounds were tested for utilizing the patient's Melanoma Cancer stem Cell Cultures established with Celprogen's Media and ECM. Among the 300 compounds tested Adria, Taxol, DTIC, Thalidomide, Paclitaxel, Abraxane, Ertibux and Velcade were not effective against Melanoma Cancer Stem cell (CSC). We were able to show 3 compounds that were effective against Melanoma CSC targeting selected pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4371. doi:10.1158/1538-7445.AM2011-4371

Retaining cell–cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer

Primary culture of the cancer cells from patients' tumors can provide crucial information of individual tumors, yet the technology has not been optimized until now. We developed an innovative culture method for primary colorectal cancer cells, based on the principle that cell-cell contact of cancer cells was maintained throughout the process. When tumor tissue was dissociated into cell clusters, in which cell-cell contact was retained, they rapidly formed spheroids that we termed cancer tissue-originated spheroids (CTOSs). CTOSs of colorectal cancer consisted of highly purified and viable cancer cells, and they were prepared with high efficiency. In immunodeficient mice, CTOSs formed xenograft tumors that retained the features of the parental tumors. Moreover, CTOSs were able to be cultured and expanded in vitro using a 3D culture system and stem cell culture medium. This method allowed evaluation of chemosensitivity and signal pathway activation in cancer cells from individual patients. Easy preparation and culture of pure primary cancer cells provides an innovative platform for studying cancer biology and developing personalized medicine.

Chondrogenesis from Human Placenta-Derived Mesenchymal Stem Cells in Three-Dimensional Scaffolds for Cartilage Tissue Engineering

Human placenta-derived mesenchymal stem cells (hPMSCs) represent a promising source of stem cells. The application of hPMSCs in cartilage tissue engineering, however, was less reported. In this study, hPMSCs were grown in a three-dimensional (3D) environment for cartilage tissue formation in vitro. To select proper scaffolds for 3D culture of mesenchymal stem cells (MSCs), rat adipose-derived MSCs were initially employed to optimize the composition and condition of the 3D environment. The suitability of a poly(D,L-lactide-co-glycolide) (PLGA) precision scaffold previously developed for seeding and culture of primary chondrocytes was tested for MSCs. It was established that MSCs had to be embedded in alginate gel before seeded in the PLGA precision scaffold for cartilage-like tissue formation. The inclusion of nano-sized calcium-deficient hydroxyapatite (nCDHA) and/or a recombinant protein containing arginine-glycine-aspartate (RGD) into the alginate gel enhanced the chondrogenesis for both rat adipose-derived MSCs and hPMSCs. The amount of extracellular matrix such as glycosaminoglycan and type II collagen accumulated during a period of 21 days was found to be the greatest for hPMSCs embedded in the alginate/nCDHA/RGD gel and injected and cultivated in the precision scaffold. Also, histological analyses revealed the lacunae formation and extracellular matrix production from the seeded hPMSCs. Comparing human bone marrow-derived MSCs (hBMSCs) and hPMSCs grown in the previous composite scaffolds, the secretion of glycosaminoglycan was twice as higher for hPMSCs as that for hBMSCs. It was concluded that the alginate/nCDHA/RGD mixed gel in the aforementioned system could provide a 3D environment for the chondrogenesis of hPMSCs, and the PLGA precision scaffold could provide the dimensional stability of the whole construct. This study also suggested that hPMSCs, when grown in a suitable scaffold, may be a good source of stem cells for building up the tissue-engineered cartilage.

Retracted: Long-Term Maintenance of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells in Suspension

Traditionally, undifferentiated pluripotent human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) have been expanded as monolayer colonies in adhesion culture, both in the presence or absence of feeder cells. However, the use of pluripotent stem cells poses the need to scale-up current culture methods. Herein, we present the cultivation of 2 hESC lines (Royan H5 and Royan H6) and 2 hiPSC lines (hiPSC1 and hiPSC4) as carrier-free suspension aggregates for an extended period of time. The cells proliferated over multiple passages kept a stable karyotype, which successfully maintained an undifferentiated state and pluripotency, as determined by marker expressions in addition to in vitro spontaneous and directed differentiation. Additionally, these cells can be easily frozen and thawed without losing their proliferation, karyotype stability, and developmental potential. Transcriptome analysis of the 3 lines revealed that the adherent culture condition was nearly identical to the suspension culture in Royan H5 and hiPSC1, but not in Royan H6. It remains unclear whether this observation at the transcript level is biologically significant. In comparison with recent reports, our study presents a low-cost procedure for long-term suspension expansion of hESCs and hiPSCs with the capability of freeze/thawing, karyotype stability, and pluripotency. Our results will pave the way for scaled up expansion and controlled differentiation of hESCs and hiPSCs needed for cell therapy, research, and industrial applications in a bioreactor culture system.

3D culture of adult mouse neural stem cells within functionalized self-assembling peptide scaffolds.

Three-dimensional (3D) in vitro models of cell culture aim to fill the gap between the standard two-dimensional cell studies and the in vivo environment. Especially for neural tissue regeneration approaches where there is little regenerative capacity, these models are important for mimicking the extracellular matrix in providing support, allowing the natural flow of oxygen, nutrients, and growth factors, and possibly favoring neural cell regrowth. We have previously demonstrated that a new self-assembling nanostructured biomaterial, based on matrigel, was able to support adult neural stem cell (NSC) culture. In this study, we developed a new 3D cell culture system that takes advantage of the nano- and microfiber assembling process, under physiologic conditions, of these biomaterials. The assembled scaffold forms an intricate and biologically active matrix that displays specifically designed functional motifs: RGD (Arg-Gly-Asp), BMHP1 (bone marrow homing peptide 1), and BMHP2, for the culture of adult NSCs. These scaffolds were prepared at different concentrations, and microscopic examination of the cell-embedded scaffolds showed that NSCs are viable and they proliferate and differentiate within the nanostructured environment of the scaffold. Such a model has the potential to be tailored to develop ad hoc designed peptides for specific cell lines.

Modeling the Prostate Stem Cell Niche: An Evaluation of Stem Cell Survival and Expansion In Vitro

The goal of this work was to engineer a clinically relevant in vitro model of human prostate stem cells (PSCs) that could be used to interrogate the mechanisms of stem cell control. We, therefore, compared the growth potential of stem cells in 3D culture (where the conditions would favor a quiescent state) with monolayer culture that has previously been demonstrated to induce PSC division. We found a fundamental difference between cultures of primary, adult PSCs grown as monolayers compared to those grown as spheres. The first supported the expansion and maintenance of PSCs from single cells while the latter did not. In an attempt to determine the mechanisms governing stem cell control, several known stem cell activators (including IFNalpha, FGF2, anti-TGFbeta, and dihydrotestosterone) were studied. However, cell division was not observed. CD133+ cells derived from a prostate cell line did not grow as spheres from single cells but did grow from aggregates. We conclude that PSCs can be expanded and maintained in monolayer culture from single cells, but that PSCs are growth quiescent when grown as spheres. It is likely that the physical arrangement of cells in monolayer provides an injury-type response, which can activate stem cells into cycle.