Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions

Journal name:
Nature Protocols
Volume:
9,
Pages:
2354–2368
Year published:
DOI:
doi:10.1038/nprot.2014.159
Published online

Abstract

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of ​Oct-4–positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm2, where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with ​E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

At a glance

Figures

  1. Representative features of hPS cells culture systems based on passaging in cell aggregates and single-cell suspensions.
    Figure 1: Representative features of hPS cells culture systems based on passaging in cell aggregates and single-cell suspensions.

    (a,b) Colonies of hPS cells grown on feeder layers. Black arrows show thickening in the middle of the colonies. Scale bars, 200 μm. (c,d) hPS cells 24 h after plating on ​Vitronectin in too-big and normal-sized pieces, respectively. Black arrow shows differentiated area. Scale bars, 200 μm. (e,f) Bright-field images of hES cells 48 h after plating on LN-521 and E8 fragment, respectively. The cells were plated at the same density. Scale bars, 500 μm.

  2. Transfer of hPS cells from feeder layer to LN-521 substratum at Step 15.
    Figure 2: Transfer of hPS cells from feeder layer to LN-521 substratum at Step 15.

    (a) Cell aggregates obtained by cutting of undifferentiated parts of hPS cell colonies grown on feeders before plating on LN-521. Scale bar, 200 μm. (b,c) Two representative colonies 24 h after plating of the aggregates on LN-521 substratum. Scale bars, 200 μm. (d) A partially differentiated colony on LN-521 4 d after the transfer. Only one piece obtained as described in a has been plated on a LN-521–coated dish in 1 ml of NutriStem hESC XF medium. Black arrow shows the differentiated area. Scale bar, 200 μm.

  3. Bright-field images of living hPS cells plated on LN-521 at different densities at different time points (Step 6 or 9).
    Figure 3: Bright-field images of living hPS cells plated on LN-521 at different densities at different time points (Step 6 or 9).

    The pictures are taken before feeding of the cells. Many rounds of unattached cells are seen floating in the medium. Scale bar, 200 μm.

  4. Flowchart of rapid passaging of hPS cell in single-cell suspensions at Step 6.
    Figure 4: Flowchart of rapid passaging of hPS cell in single-cell suspensions at Step 6.

    (a) Monolayer of hPS cells before passaging. The cells look the same in the middle and on the rim of the well. (b) The cells during TrypLE treatment. (c,d) The cells after rinsing with medium in the middle and on the rim of the well, respectively. Scale bars, 200 μm.

  5. Routine characterization of hPS cells cultured on LN-521.
    Figure 5: Routine characterization of hPS cells cultured on LN-521.

    (a) FACS analysis (Box 3) of HS346 cells for ​Oct-4, a marker of pluripotency. The percentage of positive cells is listed in parentheses. (b) Immunostaining of HS346 cells grown on LN-521 for ​Oct-4 (green). DAPI staining is shown in blue. Scale bars, 100 μm.

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Author information

Affiliations

  1. Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, Karolinska Institute, Stockholm, Sweden.

    • Sergey Rodin &
    • Karl Tryggvason
  2. Department of Clinical Sciences, Division of Obstetrics and Gynecology, Intervention and Technology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden.

    • Liselotte Antonsson &
    • Outi Hovatta
  3. Cardiovascular and Metabolic Disorders Program, Duke-NUS (National University of Singapore), Singapore, Singapore.

    • Karl Tryggvason

Contributions

S.R. and L.A. conducted in vitro experiments with the pluripotent cells. S.R., L.A. and O.H. contributed to the planning and design of experiments and to the writing of the manuscript. K.T. planned and designed the project and contributed to the writing of the manuscript.

Competing financial interests

K.T. and S.R. are shareholders in BioLamina, AB.

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