Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells

Abstract

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to provide an infinite source of tissues for regenerative medicine. Although defined xeno-free media have been developed, culture conditions for reliable propagation of hESCs still require considerable improvement. Here we show that recombinant E8 fragments of laminin isoforms (LM-E8s), which are the minimum fragments conferring integrin-binding activity, promote greater adhesion of hESCs and hiPSCs than do Matrigel and intact laminin isoforms. Furthermore, LM-E8s sustain long-term self-renewal of hESCs and hiPSCs in defined xeno-free media with dissociated cell passaging. We successfully maintained three hESC and two hiPSC lines on LM-E8s in three defined media for 10 passages. hESCs maintained high level expression of pluripotency markers, had a normal karyotype after 30 passages and could differentiate into all three germ layers. This culture system allows robust proliferation of hESCs and hiPSCs for therapeutic applications.

Figure 1. Validation of LM-E8s for H9 hESC attachment.

(a) Schematic representations of the two LM-E8s and the original intact laminin isoforms. A 6xHis-tag and FLAG-tag were attached to the N-termini of the α and γ chains, respectively, of LM-E8s to facilitate the purification of recombinant LM-E8s. The molecular weights of individual proteins are shown in parentheses. (b) Dose–response adhesion curves of H9 hESCs on various ECMs. LM-E8s at 1.5 μg cm⁻² showed higher efficiency for supporting the adhesion of dissociated cells than did other matrices. The means of absorbance (OD570) represent the relative number of adherent cells normalized against the values at the maximum effect on Matrigel, which was arbitrarily set as 1. Error bars show the s.e.m. of five independent assays, except for vitronectin, which shows the average of duplicate assays. (c) Inhibition of integrins on LM-E8s. Completely dissociated H9 hESCs were incubated with an integrin-specific blocking antibody for 30 min in mTeSR1 medium. LM511(EQ): an inactive form of LM511-E8. Error bars indicate the s.d. of five individual assays. **P<0.05; ns, non-significant; two-way ANOVA or Tukey’s test. (d) Seeding density-dependent adhesion of H9 hESCs on LM-E8s. LM-E8s showed a higher cell adhesive activity for completely dissociated H9 hESCs than did Matrigel. Completely dissociated cells were incubated on LM-E8s in mTeSR1 medium for 6 h with or without Y-27632. Data represent the means±s.d. of three experiments. **P<0.05; ns, non-significant, two-tailed Student’s t-test.

Figure 2. LM-E8s support undifferentiated proliferation in defined medium.

(a) Distinct cell survival of H9 hESCs depending on ECMs. Data represent the numbers of live cells at 24 h after seeding 3.75 × 10⁴ cells as single cells (single) or colonies (colony). (b) Serial phase-contrast images of H9 hESCs on LM-E8s or Matrigel in mTeSR1 medium at initial attachment. Images at the bottom right show an enlarged view of enclosed boxes. Note that dissociated H9 hESCs promptly extended and reached neighbouring cells on LM-E8s, but failed cellular extension on Matrigel. See also Supplementary Movies 1–5. (c) Phase-contrast images of H9 hESCs in mTeSR1 medium on LM-E8s or Matrigel during the growth phase. (d) Short-term growth curves of H9 hESCs on LM-E8s in mTeSR1 medium. Dissociated H9 hESCs on LM-E8s proliferated and had a similar growth rate as that in conventional colony culture during logarithmic growth. Data represent the means±s.d. of three individual experiments. (e) Detection of phosphorylated proteins associated with cell adhesion by western blot analysis. Cell lysates were prepared from H9 hESCs incubated for 1 h on each substrate with or without Y-27632. Sus, dissociated H9 hESCs incubated for 1 h in suspension; FN, fibronectin; VN, vitronectin. (f) Long-term growth curves of H9 hESCs on LM-E8s in mTeSR1 medium. The numbers of seeded cells at the initial passage were converted to 1, and the counted cell numbers at each passage are shown as cumulative cell numbers. Note that dissociation culture on LM-E8s resulted in a more than 200-fold increase in cell numbers relative to conventional colony culture over 1 month. Scale bars, 200 μm.

Figure 3. Flow cytometric analysis of hESC or hiPSC lines.

Three hESC lines (H9, HES3 and KhES-1) and two hiPSC line (iPS(IMR90)-1 and 253G1) were cultured on LM-E8s after complete dissociation. These cells maintained higher expression levels of the undifferentiated markers, compared with that on Matrigel with either colony or complete dissociation culture. Undifferentiated markers: SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, GCTM2 and Tra-2-54. Differentiation marker: SSEA-1. Data were collected from cells cultured for 10 passages. The percentages of positive cells are indicated on the graph.

Figure 4. LM-E8s sustain pluripotency in defined medium.

(a) Immunostaining for markers of the three germ layers in differentiating H9 hESCs cultured on LM-E8s in mTeSR1 medium after 10 passages: ectoderm (βIII tubulin), endoderm (alpha-fetoprotein (AFP)), and mesoderm (α-smooth muscle actin (SMA)). (b) RT–PCR analysis of marker genes for the differentiation of embryoid bodies. H9 hESCs were cultured in mTeSR1 medium for 10 passages on LM-E8s after complete dissociation, or on Matrigel after colony dissociation. Cells were then allowed to form embryoid bodies for 10 days. (c) Teratomas consisting of the three germ layers developed following the transfer of H9 hESCs cultured on LM-E8s after 25 passages in mTeSR1 medium. Cells were injected into the testes of SCID mice. After 8 weeks, the fate of the cells was analysed. Haematoxylin and eosin staining showed differentiation into various tissues including neuroepithelium (ectoderm), intestinal epithelium (endoderm) and cartilage (mesoderm). Scale bars, 200 μm.

Figure 5. LM-E8s sustain the pluripotency in xeno-free TeSR2 medium.

(a) Phase-contrast images of H9 hESCs. Dissociated H9 hESCs in TeSR2 medium reformed clusters (24 h) and actively proliferated (72 h). (b) Flow cytometric profiles of H9 hESCs cultured on LM-E8s for 34 passages after complete dissociation, or on Matrigel for 30 passages after colony dissociation. H9 hESCs maintained expression of markers of the undifferentiated state. Numbers indicate percentages of cells that were positive for surface markers. (c) RT–PCR analysis of the expression of pluripotency marker genes in H9 hESCs cultured in TeSR2 medium. H9 hESCs cultured for 30 passages on LM-E8s after complete dissociation maintained high-level expression of pluripotency marker genes, while differentiation lineage marker genes were suppressed. Embryoid bodies derived from H9 hESCs cultured as colonies in mTeSR1 medium were used as the control to assess differentiation. (d) Teratomas consisting of the three germ layers developed from H9 hESCs cultured on LM-E8s for 34 passages in TeSR2 medium after complete dissociation. Cells were injected into the testes of SCID mice. After 8 weeks, the fate of the cells was analysed. Haematoxylin and eosin staining showed differentiation into various tissues including neuroepithelium (ectoderm), intestinal epithelium (endoderm) and cartilage (mesoderm). Scale bars, 200 μm.