Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them

Abstract

Background: Human embryonic stem cells (hESCs) may provide an invaluable resource for regenerative medicine. To move hESCs towards the clinic it is important that cells with therapeutic potential be reproducibly generated under completely defined conditions.

Methodology/principal findings: Here we report a four-step scalable process that is readily transferable to a Good Manufacture Practice (GMP) facility for the production of functional dopaminergic neurons from hESCs for potential clinical uses. We show that each of the steps (propagation of ESC-->generation of neural stem cells (NSC)-->induction of dopaminergic precursors-->maturation of dopaminergic neurons) could utilize xeno-free defined media and substrate, and that cells could be stored at intermediate stages in the process without losing their functional ability. Neurons generated by this process expressed midbrain and A9 dopaminergic markers and could be transplanted at an appropriate time point in development to survive after transplant.

Conclusions/significance: hESCs and NSCs can be maintained in xeno-free defined media for a prolonged period of time while retaining their ability to differentiate into authentic dopaminergic neurons. Our defined medium system provides a path to a scalable GMP-applicable process of generation of dopaminergic neurons from hESCs for therapeutic applications, and a ready source of large numbers of neurons for potential screening applications.

Figure 1. Generation of NSCs from hESCs adapted to defined medium.

hESC line I6 at passage 42 was adapted to a chemically defined medium StemPro. (A–B) Morphology (A) and expression of the pluripotent marker Oct4 (B) in hESCs that were cultured in StemPro for 28 passages. (C–F) Generation of NSCs in defined conditions. Nestin⁺ neural tube-like rosette structures were formed in the center of the ESC colonies after 12–14 days of differentiation (C–D). A monolayer of homogeneous NSCs expressed Sox1 and nestin (E–F).

Figure 2. NSCs cultured in xeno-free defined medium for a prolonged period remain to be multipotent.

(A) NSCs propagated in defined conditions for 15 passages retained NSC identity as evident by expressing Sox1 and nestin. (B–C) NSCs can be frozen and thawed and maintained the capacity to differentiate into neurons (B), oligodendrocytes (C) and astrocytes (D).

Figure 3. NSCs from prolonged culture in defined conditions can be efficiently differentiated into midbrain dopaminergic neurons.

(A–E) Efficient differentiation into midbrain dopaminergic neurons by PA6-CM as shown by immunocytochemistry. The majority of the cells expressed β-III-tubulin and TH after 4 weeks of differentiation in PA6-CM (A–B). Co-expression of midbrain and A9 markers in TH⁺ dopaminergic neurons: Lmx1a (C), VMAT (D) and Girk2 (E). (F) Differential expression of dopaminergic markers in several stages of differentiation (NSC, dopaminergic precursors and dopaminergic neurons) by quantitative PCR. All the examined markers were up-regulated in dopaminergic populations compared to NSCs.

Figure 4. Four-step differention into midbrain dopaminergic neurons in a completely defined medium.

(A) A schematic diagram of the four-step (ESC→propagation and storage of NSC→induction of a midbrain dopaminergic precursor population→maturation of the precursor to dopaminergic neurons) differentiation protocol for generation of dopaminergic neurons in completely defined media. (B–D) Defined medium adapted NSCs differentiated into dopaminergic neurons in defined media.

Figure 5. NSCs differentiated into functional dopaminergic neurons in vivo.

Day 20 cells (after the NSC stage) were transplanted into the striatum of rats. Differentiation and survival of donor-derived dopaminergic neurons are seen in striatum two (A–D) and seven (E–H) weeks post of transplantation. Transplantation of day 20 cells in 6-OHDA rats showed a subset of human dopaminergic neurons, which survived eight weeks post of graft (I–L). Rotary tests showed amelioration of behavioral deficits in PD rats transplanted with donor-derived dopaminergic neurons (M).

Figure 6. Completely serum- and xeno-free defined culturing of hESCs and NSCs.

Morphology and expression of Oct4 (A) in hESCs that were cultured in xeno-free medium (hKSR with growth factors) for 7 passages. NSCs derived from xeno-free cultured hESCs expressed nestin and Sox1 (B–C) and could differentiate into dopaminergic neurons (D).

Figure 7. Generation of dopaminergic neurons from serum-free derived hESCs.

NSCs could be generated from hESC line derived under serum-free and minimal xeno component exposure conditions as efficient as from defined medium adapted hESCs. Expression of nestin in NSCs (A) and differentiation into dopaminergic neurons (B).