Neural induction from ES cells portrays default commitment but instructive maturation

Abstract

The neural induction has remained a debatable issue pertaining to whether it is a mere default process or it involves precise instructive cues. We have chosen the embryonic stem (ES) cell model to address this issue. In a devised monoculture strategy, the cell-cell interaction availed through optimum cell plating density could define the niche for the attainment of efficient in vitro neurogenesis from the ES cells. The medium plating density was found ideal in generating optimum number of progenitors and also yielded about 80% mature neurons in a serum free culture set up barring any exogenous inducers. We could also demarcate and quantify the neural stem cells/progenitors among the heterogeneous cell population of differentiating ES cells using nestin intron II driven EGFP expression as a tool. The one week post-plating was determined to be the critical time window for optimum neural progenitor generation from ES cells that helped us further in purifying these cells and in demonstrating their proliferation and multipotent differentiation potential. Seeding cells at varying densities, we could decipher an interesting paradoxical scenario that interlinked both commitment and maturation with the initial plating density having a vital influence on neuronal maturation but not specification and the secretory factors were apparently playing a key role during this process. Thus it was comprehended that, the neural specification was a default process independent of exogenous factors and cellular interaction. Conversely, a defined number of cells at the specification stage itself seemed critical to provide an auto-/paracrine means of signaling threshold for the maturation process to materialize.

Figure 1. The neural differentiation was more pronounced in KO medium in both monolayer and EB cultures.

The ES cells line, D3 when grown in a monolayer culture in serum containing medium for 2 days followed by 6 days (8 dpp) in KO (A) or KR (B) medium displayed EB like morphology with cell migration in a centrifugal fashion and processes sprouting (arrow) from the cell dense central part (arrowhead). RA expedited the neural differentiation process that was often associated with extensive interconnections and lattice formation. (B, C). The differentiation was comparable in KO medium at 13 dpp with (E) or without (D) RA exposure. The EBs generated by hanging drop and exposed to RA also exhibited extensive neural differentiation (d7+7) upon plating when cultured in KO medium (F). The ES cells displayed a cellular mat (G) when cultured in DM in a monolayer without RA exposure (13 dpp), while in presence of RA (H) had cells with neural morphology. Scale: 30 µM.

Figure 2. The influence of medium on neural differentiation profile in nestin transgenic ES cell clones.

The differentiation pattern in nes-EGFP cells as seen in bright field and fluorescence at 8 dpp and 10 dpp in KO (A,B and C,D) and FBS containing (E,F and G) medium respectively. The neural differentiation was promoted in KO medium (A–D) compared to that in serum containing one (E–G). RA expedited the process with better differentiation in respective media (B, D and G). While the neural progenitors were EGFP⁺, the mature ones with extensive processes were EGFP⁻ (H) corresponding with the endogenous nestin expression pattern. Scale: 30 µM.

Figure 3. The neural differentiation and immuno-cytochemical characterization.

The expression of early neural markers Sox2 and nestin was detected in an overlapping fashion at various time periods during differentiation (A–C) with Sox2 showing nuclear localization (Dapi⁺: A). Extensive neuronal differentiation was demonstrated by Map2 stained cells (D) including 5-HT⁺ serotonergic (E) and TH⁺ dopaminergic (F) neuronal subtypes. This 2D culture strategy also yielded GFAP⁺ astrocytes (G) and O4⁺ oligodendrocytes (H). The Map2⁺ cells were also synaptophysin+ (J; red: synaptophysin, blue: Dapi). Scale: 40 M except D and G (10 µM).

Figure 4. The influence of plating density on neural progenitor generation and differentiation in nes-EGFP ES cells.

The cells plated at 2 K density showed EGFP⁺ neural progenitors in KO (A) or KR (B) medium, however, without any apparent differentiating neural cells at 8 dpp. The cells plated at medium (60 K; C, D) and high (130 K; E, F) densities showed prominent neural differentiation at the said time period. RA exposure (D) and (F) at the respective densities facilitated pronounced neural sprouting. Figures are shown in bright field and fluorescence. Scale: 30 µM.

Figure 5. Immunophenotyping of nes-EGFP cells during differentiation.

The nes-EGFP cells expressing EGFP during differentiation also expressed nestin and Sox1 as seen at 2 dpp (A). Contrary to nestin, the Sox1 expression window was transient with enhanced expression at 4 dpp (B) that became negligible by 7 dpp (C). The nes-EGFP cells exhibited rosette like structures during differentiation with co-localization of EGFP and endogenous nestin (D). The Map2⁺ differentiating neurons were EGFP⁻, however remained associated with EGFP⁺ neural progenitors (E). The histogram represents the Sox1 population during differentiation as quantified by flowcytometry (F). Scale: 40 µM.

Figure 6. The temporal profile of neuronal differentiation in nes-EGFP cells.

A few Map2⁺ neurons were detected as early as 6 dpp in cells seeded at medium density and cultured in KO (A) or KR (B) medium. The Map2 expression was extensive at 9 dpp in cells cultured in either KO (C, E) or KR (D, F) medium and plated at medium (C, D) or high (E, F) densities respectively. The cells plated at low density, however, had attenuation in neuronal differentiation at the detected time point (G; 9 dpp). Scale: 40 µM.

Figure 7. The differentiation of human ES cells into neurons.

An undifferentiated human ES cell colony grown on mitotically inactivated mouse embryonic fibroblast monolayer (A). The human ES cells were differentiated into neurons (d25) in the KR medium showing Map2 positive staining (B). Some of the Map2⁺ neurons (C) were also TH⁺ (D) indicating their dopaminergic neuronal subtype status. Scale: 30 µM.

Figure 8. The RT-PCR analysis for secretory factors during neurogenesis in cells plated at varying densities.

(A): BMP4, FGF4, Noggin expression status in undifferentiated ES cells and during various days of differentiation in cells plated at medium (50 K) density. β-actin gene served as a housekeeping positive control. (B): The expression status of BMP4, FGF4, Wnt3a, Wnt5a, Wnt8a, Wnt8b at d6 and d8 of differentiation in cells plated at varying densities. The bottom row shows the housekeeping β-actin control with (+) and without (−) reverse transcriptase in the reactions. (C): The semi-quantitative (densitometry: Wnt 3a) and quantitative (Wnt 5a, Wnt 8a) detection of Wnt transcripts relative to β-actin, keeping the relative expression count at 50 K density as 1. Data are represented as mean+/−SEM (n = 4–5) with P<0.05 (Wnt5a) and <0.01 (Wnt8a) using paired t- test for comparing the transcripts between 2 K and 50 K groups.

Figure 9. The quantification of EGFP⁺ neural progenitors by flowcytometry.

(A): The nes-EGFP cells cultured in monolayer generated bright EGFP⁺ neural progenitors (M2 population) with maximum number being detected at d7. The M1 population included also the weak EGFP⁺ cells representing the differentiating population. (B): The bar diagram shows the neural progenitor generation profile comparing the EGFP population at the M2 window at different time points during differentiation keeping the ES cells value as 1 (n = 4–6). The EGFP⁺ cells peaked during 6–9 dpp, the optimum time window for efficient neural progenitor generation during differentiation followed by a decrease in the second week. (C): The bar diagram shows the influence of plating density and RA exposure of cells on EGFP⁺ neural progenitor generation at 7 dpp. Data are represented as mean+/−SEM (n = 4–7).

Figure 10. The differentiation profile of neural progenitors purified by MACS.

The nes-EGFP cells 8 dpp were subjected to MACS, the A2B5⁺ eluent (A–D) and SSEA1⁻ flow through (G) were collected and grown in culture. The A2B5⁺ cells cultured in either DM (A: 6 dpp; B: 10 dpp) or KO medium (C: 6 dpp; D: 10 dpp) exhibited proliferation and differentiation with better differentiation in KO (D) that showed both Map2⁺ neurons (E) and GFAP⁺ astrocytes (F). Similarly the SSEA1⁻ cells when cultured in KO medium (G) underwent differentiation into Map2⁺ neurons (H) and GFAP⁺ astrocytes (J). Scale: 30 µM.