B-27tm Supplement (50x) Serum Free

[Tyler Bannowsky]

NCS21Neuronal Supplement is a serum-free supplement for neuronal cell cultures. It is an optimized and modified formulation of B27 Supplement (B27 is a registered trademark of Life Technologies Corporation).

Suitable applications include, e.g., differentiation of ES cells into neuron lineage (neuron and astrocytes), differentiation of neuronal stem cells into astrocytes and neurons, optimal growth and long-term survival of rat hippocampal neurons (fetal and adult), survival of neurons from embryonic rat striatum, substantia nigra, septum and cortex, and neonatal rat cerebellum (fetal and adult).

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In vitro differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) represents a key step towards somatic cells of lung, liver and pancreas. For future clinical applications, mass production of differentiated cells at chemically defined conditions and free of xenogeneic substances is envisioned. In this study we adapted our previously published two-dimensional (2D) DE induction protocol to three-dimensional (3D) static suspension culture in the absence of the xenogeneic extracellular matrix Matrigel. Next, fetal calf serum and bovine serum albumin present in the standard medium were replaced by a custom-made and xeno-free B-27. This yielded in a chemically defined and xenogeneic-free 3D culture protocol for differentiation of hPSCs into DE at efficiencies similar to standard 2D conditions. This novel protocol successfully worked with different hPSC lines including hESCs and hiPSCs maintained in two different stem cell media prior to differentiation. DE cells obtained by our novel BSA-free 3D protocol could be further differentiated into PDX1- or NKX6.1-expressing pancreatic progenitor cells. Notably, upon DE differentiation, we also identified a CXCR4+/NCAM+/EpCAMlow cell population with reduced DE marker gene expression. These CXCR4+/NCAM+/EpCAMlow cells emerge as a result of Wnt/beta-catenin hyperactivation via elevated CHIR-99021 concentrations and likely represent misspecified DE.

Human pluripotent stem cells (hPSCs) possess an unlimited proliferative potential and can be differentiated into all somatic cell types. Owing to these properties they represent an attractive cell source for cell replacement therapies, pharmacological studies on defined somatic cell types and basic research such as the study of human development1.

To the best of our knowledge, no chemically defined and xenogeneic-free approach for DE differentiation of hPSCs as free-floating suspension culture spheroids in 3D culture has been published until today. Thus, we established static 3D conditions for DE differentiation of hPSCs in the absence of xenogeneic scaffolds and media supplements based on our previously published protocol4. All xenogeneic components were successively replaced or removed without negative effects compared to the original protocol. 3D conditions also supported further differentiation of DE cells towards PDX1-positive pancreatic progenitors (PPs)27 using an adapted, chemically defined and BSA-free media formulation. Additionally, we noticed that DE differentiation conditions gave rise to a subpopulation of CXCR4+ that was also positive for NCAM. These cells could be associated with a decreased expression of important DE marker genes suggesting that DE differentiation protocols should be optimized towards low NCAM-positivity.

Overview of media used for DE differentiation of hPSCs. DE differentiation of hPSCs was performed according to the CA-A approach4, but with different basal media and supplements. Media formulations were changed step-wise. First, FCS was replaced by a modified B-27 (mB-27) to obtain chemically defined conditions. Second, advanced RPMI 1640 was replaced by MCDB131 or RPMI 1640 for BSA-free conditions. Third, mB-27 was replaced by mB-27XF to obtain a fully xenogeneic-free condition.

Differentiation according to the STD protocol was performed in the presence of FCS. In order to establish a chemically defined (CD, Fig. 1) condition, FCS was replaced by our custom made serum-free media supplement called modified (m)B-27 (Table 1)28. This mB-27 is related to the commercially available B-27TM but lacks insulin and BSA. The CD condition was tested in 2D (CD-2D) and 3D (CD-3D) culture for its capability to support DE differentiation of hPSCs. The proportion of CXCR4+ cells and proliferation under CD-2D and STD-2D conditions were comparable for HES3 and HUES8 cells (Figs 3A,B and S1A,B). As shown for HES3, this was independent of the hPSC maintenance medium (Figs 3A and S1A). Similar, during small scale 3D culture, the CD condition supported DE differentiation of HES3 comparable to the STD condition (Figs 3C and S1C). Gene expression of typical endodermal and pluripotency marker genes during CD-3D and STD-3D conditions were comparable (Fig. 3D). Similar results in 3D culture were also obtained for the human induced pluripotent stem cell (hiPSC) line hCBiPSC2 (Fig. 3E,F). Taken together, replacement of FCS by mB-27 enabled chemically defined differentiation of hPSCs in 2D and 3D culture without negative effects on the DE induction.

Interestingly, we observed in BF and XF conditions less NCAM+ cells within the CXCR4+ population in RPMI medium at lower CHIR concentrations. Typical DE genes were significantly reduced in CXCR4+/NCAM+/EpCAMlow compared with CXCR4+/EpCAM+ cells. Thus, these cells might represent a falsely specified endoderm subpopulation. CXCR4+/NCAM+/EpCAMlow cells strongly expressed MIXL1, whereas GSC was reduced. This indicates that this population could be stuck in a primitive streak pattern or was partially committed towards a mesodermal cell fate. Accordingly, Kempf et al. recently proposed a model, in which the concentration of CHIR and its duration affects primitive streak-like patterning along the anterior-posterior axis influencing cell fate decisions towards DE, cardiac mesoderm or presomitic mesoderm45. Along with Lian et al., who reported that BSA reduces the activity of CHIR during mesodermal differentiation46 and the issue that NCAM-positivity in the CXCR4+ population increased in a CHIR concentration-dependent manner, the CHIR concentration should be carefully determined for BSA-free differentiation conditions. Furthermore, our data suggests NCAM-positivity as a negative marker for DE quality.

DE cells obtained by the newly established BF condition for DE formation in 2D and 3D culture could be further differentiated into PDX1+ pancreatic progenitor (PP) under BSA-free conditions adapted from our recently published protocol27 and into multipotent pancreatic NKX6.1+ progenitor cells according to the Kieffer-protocol33. Actually, 3D culture was shown to be superior to 2D culture for further maturation of DE cells into PDX1+ PP cells and subsequently into insulin producing cells24,47,48, which underlines the importance of scalable 3D cultures. It can be expected that the microenvironment within these 3D aggregates mimics the in vivo distribution and accessibility of morphogens and nutrients and thus may enhance maturation of differentiated progeny49,50. In line with our findings, Pagliuca et al. reported the efficient differentiation of 3D suspension culture spheroids of hPSCs into PDX1+ PP cells and into functional pancreatic beta cells24. In the future fully defined and xenogeneic-free differentiation protocols for the generation of pancreatic beta cells will be required.

As our mB-27 still contained a small amount of bovine-derived proteins (superoxide dismutase and catalase), we also omitted these proteins to obtain a 100% xenogeneic-free formulation designated mB-27XF. This mB-27XF supported DE differentiation to a similar extent as the CD, BF or STD condition, which is in concordance with Wang et al., who reported that antioxidants in B-27TM were redundant during DE differentiation of hPSCs16.

This work has been supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy, EXC 62/2). FFRB was funded by the DFG (FOR2509 BU 2920/2-1 and BU 2920/3-1). ON and UD were funded by the DFG (NA 1285/2-1). UD was funded by the internal program (HiLF) of the Hannover Medical School. The skilful technical assistance of J. Kresse, M. Funck and A. Oberbeck is gratefully acknowledged. We would like to acknowledge the assistance of the Cell Sorting Core Facility of the Hannover Medical School supported by the Braukmann-Wittenberg-Herz-Stiftung and the DFG.

In this study, SC medium (1701, ScienCell, Carlsbad, CA) and NeurobasalTM Medium (21103049, Gibco), with the respective basal glucose concentrations 5.5 mM and 25 mM, were used. The complete neurobasal medium was prepared by supplementing 2% B-27TM Supplement (50X) (17504044, Gibco) and 0.5 mM GlutaMAXTM Supplement (35050061, Gibco). The complete SC medium was prepared by adding 25 mL of fetal bovine serum (FBS), 5 mL of Schwann Cell Growth Supplement, and 5 mL of penicillin/streptomycin solution. Hyperglycemic media was prepared by adding glucose (47829, Sigma-Aldrich, Singapore) to these complete media. In particular, the glucose level of the SC medium was adjusted to 10, 30, and 60 mM and that of the Neurobasal Medium was adjusted to 35, 45, and 60 mM to produce hyperglycemic insults. These media were used at the cell time of seeding. For culture experiments lasting for more than 2 days, media was changed every 2 days to maintain the hyperglycemic condition during the experiments. For co-culture studies, SCs (ScienCell) were seeded into a 96-well plate at the density of 5,000 cells/well (for coculture in hyperglycemic insults alone) or 3,000 cells/well (for coculture in hyperglycemic insult supplemented with insulin) and induced to the mature phase in the complete SC medium supplemented with vitamin C (50 μg/mL) for 4 days prior to seeding neurons on top of the SC layers. The complete Neurobasal Medium with the assigned glucose concentrations was used for co-culture after seeding the neurons. All hyperglycemia cultures were performed in a randomized manner.

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