Xeno-free culture of pluripotent stem cells in NutriStem® hPSC XF medium

[fa icon="calendar"] Feb 5, 2018 5:58:00 AM / by Robert Annand

Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are a key component of the growing field of regenerative medicine. Use of culture conditions free of animal products (xeno-free) is essential for PSC projects that are destined for the clinic. NutriStem hPSC XF medium (01-0005) and iMatrix-511 recombinant human laminin-511 E8 fragments (NP8920911) form an ideal system for xeno-free, feeder culture of human PSCs.

 

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NutriStem® hPSC XF Medium

nutristem_boxNutriStem® hPSC XF Medium is a defined, xeno-free, serum-free culture medium that is made to GMP specifications and designed to support the propagation and expansion of human ESCs and iPSCs in a feeder-free culture system. NutriStem contains low concentrations of growth factors such as FGF-basic, providing a simplified medium to maintain the cells full differentiation potential. NutriStem has been used to support a wide variety of iPS differentiation protocols, including cardiomyocytes[1], neurons[2], hepatocytes[3], and endothelial progenitor cells (EPCs)[4]. A Drug Master File for NutriStem hPSC XF medium is on file with the US FDA to support clinical applications.

 

 NutriStem_Jan2018_fig1-2

Figure 1: NutriStem® hPSC XF Medium enables excellent proliferation of undifferentiated hES and hiPS cells. Proliferation of H1 hES cells cultured in Corning Matrigel-coated 96-well plates in NutriStem XF/FF Medium and the leading competing medium for feeder-free culture. Medium was changed and proliferation was assessed every 24 hours in culture.

 

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Figure 2: iPSCs cultured in NutriStem hPSC XF medium 

 

iMatrix-511

mxome_kit_boxRecombinant Laminin-511 E8 Fragments is a chemically-defined, xeno-free human laminin substrate for cell growth. Laminin-511 E8 fragments have been shown to support greater adhesion of human ESCs than Corning® Matrigel®, fibronectin, or vitronectin[5], presumably through interaction with a6b1 integrin in PSCs. No karyotypic abnormalities were observed after at least 20 passages of PSCs on laminin-511 E8 fragments. iMatrix-511 supports high-efficiency reprogramming of a variety of target cells to iPSCs using REPROCELL’s Stemgent® StemRNA™ 3rd Gen Reprogramming technology (00-0076).

 Laminin-511-E8_fragment-1

Figure 3: Structure of Laminin-511 E8 fragment.

 

NutriStem_Jan2018_fig4

Figure 4: Adhesion of H9 ESCs to iMatrix-511 coated plates surpasses that of other popular coatings. Adhesion was measured 24 hr after plating, by washing and staining staining with crystal violet. Data from reference 5.

 

A powerful system for culturing human iPSCs and ESCs

Using NutriStem and iMatrix-511 together gives you a powerful system for feeder-free, xeno-free culture of human iPSCs and ESCs. Cells from skin (fibroblasts) as well blood (endothelial progenitor cells; EPCs) and urine (urine-derived epithelial cells) are readily reprogrammed using the StemRNA 3rd Gen technology (00-0076), and the iPSCs maintain pluripotency, as shown by ICC and in vitro differentiation, even after extensive passaging. This system provides a robust, xeno-free culture system for human PSCs.

 

Human-iPSCs-from-EPCs

Figure 5. Human iPSCs derived from EPCs were cultured in NutriStem hPSC XF medium on iMatrix-511.  At passage 6 the cells were stained with DAPI and anti-SSEA-4 antibody.

 

iPSCs-NutriStem-iMatrix-511-early-endoderm iPSCs-NutriStem-iMatrix-511-neuronal-cells iPSCs-NutriStem-iMatrix-511-cardiomyocytes

Figure 6: Human iPSCs at passage 8 in NutriStem medium on iMatrix-511 were differentiated in vitro and stained for specific markers.
Left: Early endoderm: stained with DAPI (blue) and anti-AFP (red).
Center: neuronal cells: stained with anti-nestin (red) and anti-β-tubulin (green).
Right: Cardiomyocytes: stained with DAPI (blue) and anti-troponin T (red).

 

References

  1. Jacquet L; Neueder A; Földes G; Karagiannis P; Hobbs C; Jolinon N; Mioulane M; Sakai T; Harding SE; Ilic D. Three Huntington’s disease specific mutation-carrying human embryonic stem cell lines have stable number of CAG repeats upon in vitro differentiation into cardiomyocytes. PLoS ONE 10(5):e0126860 (2015)

  2. Schwab AJ; Ebert AD. Neurite aggregation and calcium dysfunction in iPSC-derived sensory neurons with Parkinson’s disease related LRRK2 G2019S mutation. Stem Cell Reports 5(6): 1039-52 (2015).

  3. Tolosa L; Caron J; Hannoun Z; Antoni M; Lopez S; Burks D; Vicente Castell J; Weber A; Gomez-Lechon M-J; Dubart-Kupperschmitt A. Transplantation of hESC-derived hepatocytes protects mice form liver injury. Stem Cell Res Therapy 6:246 (2015).

  4. Nguyen MTX; Okina E; Chai X; Tan KH; Hovatta O; Ghosh S; Tryggvason K. Differentiation of human embryonic stem cells to endothelial progenitor cells on laminins in defined and xeno-free system. Stem Cell Reports 7(4): 802-16 (2016).

  5. Miyazaki T; Futaki S; Suemori H; Taniguchi Y; Yamada M; Kawasaki M; Hayashi M; Kumagai H; Nakatsuji N; Sekiguchi K; Kawase E. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nature Commun. 3:1236 (2012).

  6. Eminli-Meissner S; Yi K; Moon J-I; Poleganov M; Beissert T; Sahin U; Huang C; Morell N Rana A; Hamilton B. Use of non-modified RNAs for the derivation of clinically-relevant iPS cell lines from human blood, urine and skin cells using GMP-compliant reagents. A poster presented at the 2016 ISSCR Annual meeting, San Francisco.

Topics: Stem Cells, Human Tissues, Drug Discovery

Robert Annand

Written by Robert Annand

Robert R. Annand, Ph.D.
Senior Technical Product Manager
REPROCELL USA, Inc.