Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells

Abstract

We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR™1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel™-coated substrates. Mechanistically, it was resolved that bovine serum albumin (BSA) in the mTeSR™1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g., peptides, proteins, or Matrigel™) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion.

Figure 1. Hydrogel surfaces of aminopropylmethacrylamide (APMAAm) were synthesized and characterized

(a) Survey data from XPS analysis of APMAAm-modified TCPS. (b) Survey data from XPS analysis of TCPS.

Figure 2. Pluripotency of H1s and H9-hOct4-pGZs was maintained for 10 passages on APMAAm

Representative phase contrast images of H1s on (a) APMAAm and (b) Matrigel^™ -coated substrates at p8 (scale bar = 25μm). Representative confocal image stacks at p10 of H1 colonies on APMAAm stained with (c) Dapi, (d) Oct-4, and (e) SSEA-4; compared with H1 colonies on Matrigel^™ stained with (f) Dapi, (g) Oct-4, (h) SSEA-4 (scale bar = 10μm). (i) H9-hOct4-pGZ cell attachment at p1 and p22 onto APMAAm and Matrigel^™ normalized to Matrigel^™. (j) H1s and H9-hOct4-pGZs had a normal karyotype after p10 on APMAAm surfaces (H1 karyotype shown). (k) H9-hOct4-pGZ proliferation at p1 on APMAAm and Matrigel^™ surfaces (normalized to Matrigel^™ d1). (l) H9-hOct4-pGZ proliferation at p22 on APMAAm and Matrigel^™ (normalized to Matrigel^™ d1). Quantitative immunostaining of (m) H1s and (n) H9-hOct4-pGZ for Oct4, SSEA-4, and TRA-1-60 after p10 on APMAAm and Matrigel^™. (o) Quantitative RT-PCR results for puripotent markers Oct4, Sox2, and Nanog at p10 (H1).

Figure 3. H9-hOct4-pGZ cells were differentiated into EBs after 12 passages on APMAAm and Matrigel^™

Phase contrast images of Day 11 EBs from cells originally cultured on (a) APMAAm and (b) Matrigel^™ surfaces. Immunostaining for germ layer markers on day 40 for EBs derived from cells cultured on APMAAm surfaces: (c) Desmin (endoderm); (d) Smooth Muscle Actin (SMA; mesoderm); (e) β-Tubulin III (ectoderm); and, on Matrigel^™: (f) Desmin; (g) SMA; and (h) β-Tubulin III (scale bars = 10 μm).

Figure 4. Protein adsorption to APMAAm from mTeSR^™1

Kinetics of protein adsorption from mTESR^™1 complete media onto the APMAAm as determined by QCM-D. Initially APMAAm-modified sensor crystals were baselined in DPBS, where no adsorption to the surface occurs. Next, complete mTESR^™1 media was introduced into the chamber and resulted in a decrease in (a) frequency (ΔF) of the crystal (corresponding to an increase in adsorbed mass) and an increase in the (b) dissipation factor (D). After rinsing, the final mass of the adsorbed film was 460 ng/cm². QCM-D measurements of the (c) ΔF and (d) ΔD with the adsorption of a layer of BSA onto the APMAAm from incomplete mTeSR^™1 media supplemented with only BSA. Subsequent introduction of complete mTeSR^™1, followed by PBS, leads to a stable adsorbed protein film of 810 ng/cm².

Figure 5. BSA adsorption to APMAAm surfaces

(a) H9-hOct4-pGZ attachment to APMAAm surfaces after 24 h in complete mTeSR^™1 and incomplete mTeSR^™1 supplemented with: BSA, TGF-β or bFGF, (normalized to complete mTeSR^™1). The addition of BSA to imTeSR^™ 1 led to more than four times the number of hESCs attached compared to the imTeSR^™ 1 with either bFGF or TGF-β. (b) A QCM-D was employed to record the adsorption of BSA from solutions at different concentrations in PBS onto APMAAm surfaces. The footprint size of BSA molecules at τ-75 was calculated, where larger footprint sizes resulted from lower concentration solutions indicating spreading. When BSA was adsorbed from incomplete mTeSR^™1 media, it adsorbed in the smallest footprint size of ~ 16nm², which indicated no spreading.