Production and validation of a good manufacturing practice grade human fibroblast line for supporting human embryonic stem cell derivation and culture

Abstract

Introduction: The development of reproducible methods for deriving human embryonic stem cell (hESC) lines in compliance with good manufacturing practice (GMP) is essential for the development of hESC-based therapies. Although significant progress has been made toward the development of chemically defined conditions for the maintenance and differentiation of hESCs, efficient derivation of new hESCs requires the use of fibroblast feeder cells. However, GMP-grade feeder cell lines validated for hESC derivation are not readily available.

Methods: We derived a fibroblast cell line (NclFed1A) from human foreskin in compliance with GMP standards. Consent was obtained to use the cells for the production of hESCs and to generate induced pluripotent stem cells (iPSCs). We compared the line with a variety of other cell lines for its ability to support derivation and self-renewal of hESCs.

Results: NclFed1A supports efficient rates (33%) of hESC colony formation after explantation of the inner cell mass (ICM) of human blastocysts. This compared favorably with two mouse embryonic fibroblast (MEF) cell lines. NclFed1A also compared favorably with commercially available foreskin fibroblasts and MEFs in promoting proliferation and pluripotency of a number of existing and widely used hESCs. The ability of NclFed1A to maintain self-renewal remained undiminished for up to 28 population doublings from the master cell bank.

Conclusions: The human fibroblast line Ncl1Fed1A, produced in compliance with GMP standards and qualified for derivation and maintenance of hESCs, is a useful resource for the advancement of progress toward hESC-based therapies in regenerative medicine.

Figure 1

Derivation of the clinical grade fibroblast line NclFed1A. (a) A detailed description of the Quality Management System (QMS) required for processing cells to good manufacturing practice (GMP; passaging of NclFed1A). (b) Schematic showing the general processes involved in deriving a fibroblast cell line under the control of GMP. "Upstream" includes all processes that are in place before the derivation and expansion of the fibroblast line. "Downstream" refers to biosafety and functionality testing of the line.

Figure 2

Characterization of NclFed1A as fibroblasts. (a) NclFed1A has a normal fibroblast morphology. (b) Cells were positive for HFF-Cellect, a mouse monoclonal antibody derived from mice immunized with human foreskin fibroblasts (scale bar, 50 μm). (c) Cells were positive for the fibroblast marker 5B5 required for triple-helix formation in collagen (scale bar, 50 μm). (d-f) Histograms from flow cytometry showing the proportion of cells positive for (d) CD90, (e) CD166, and (f) CD44. (g) NclFed1A had a normal male karyotype. (h) The doubling time of NclFed1A was 26.7 hours; no significant difference was found between PD0, PSB, MCB, and 10 (PD10) and 28 (PD28) cell doublings from the MCB.

Figure 3

NclFed1A supports human embryonic stem cell (hESC) derivation and culture. (a) The proportion of outgrowths and hESC lines produced by using NclFed1A and MEFS (derived from CF1 and C57Bl6 strains) as feeder cells. (b) hESCs cultured on NclFed1A for 24 passages were positive for the pluripotency markers NANOG and OCT4 (scale bar, 100 μm). (c) A greater proportion of cells expressed Tra-1-60 when cultured on NclFed1A than on MRC-5 (P < 0.05); no significant difference was noted between the expression of pluripotency markers after culture on NclFed1A, iHDFn, and iMEF. (d) The expression of pluripotency markers was similar after culture on NclFed1A at PD8, PD18, and PD28; at PD38, fewer cells expressed Tra-1-81 and SSEA4 (P < 0.05). (e) hESCs cultured on NclFed1A at P15 had normal hESC morphology. (f) hESCs cultured on NclFed1A at PD38 showed signs of differentiation (arrows).

Figure 4

High-performance liquid chromatography (HPLC) analysis showing the concentration of Neu5Gc in cells cultured in xeno-free human embryonic stem cell (hESC) medium (KOSR-XF) and xeno-containing medium (KOSR). (a) The concentration of Neu5Gc in inactivated and noninactivated NclFed1A cells cultured in KOSR-XF and KOSR medium for 2 to 6 days. (b, c) The HPLC trace of Neu5Gc concentration in noninactivated NclFed1A cultured for 6 days in (b) KOSR and (c) KOSR-XF.