Human pluripotent stem cell (PSC)-derived mesenchymal stem cells (MSCs) show potent neurogenic capacity which is enhanced with cytoskeletal rearrangement

Abstract

Mesenchymal stem cells (MSCs) are paraxial mesodermal progenitors with potent immunomodulatory properties. Reports also indicate that MSCs can undergo neural-like differentiation, offering hope for use in neurodegenerative diseases. However, ex vivo expansion of these rare somatic stem cells for clinical use leads to cellular senescence. A newer source of MSCs derived from human pluripotent stem cells (PSC) can offer the 'best-of-both-worlds' scenario, abrogating the concern of teratoma formation while preserving PSC proliferative capacity. PSC-derived MSCs (PSC-MSCs) also represent MSCs at the earliest developmental stage, and we found that these MSCs harbor stronger neuro-differentiation capacity than post-natal MSCs. PSC-MSCs express higher levels of neural stem cell (NSC)-related genes and transcription factors than adult bone marrow MSCs at baseline, and rapidly differentiate into neural-like cells when cultured in either standard neurogenic differentiation medium (NDM) or when the cytoskeletal modulator RhoA kinase (ROCK) is inhibited. Interestingly, when NDM is combined with ROCK inhibition, PSC-MSCs undergo further commitment, acquiring characteristics of post-mitotic neurons including nuclear condensation, extensive dendritic growth, and neuron-restricted marker expression including NeuN, β-III-tubulin and Doublecortin. Our data demonstrates that PSC-MSCs have potent capacity to undergo neural differentiation and also implicate the important role of the cytoskeleton in neural lineage commitment.

Keywords: Rho A kinase (ROCK); human embryonic stem cells (ESCs); human pluripotent stem cells (PSC); induced pluripotent stem cells (iPS); mesenchymal stem cells (MSCs).

Figure 1. Human pluripotent stem cell-derived mesenchymal stem cells (PSC-MSCs) express higher level of neural stem cell-associated genes than bone marrow mesenchymal stem cells (BM-MSCs)

(A) Gene expression of Nestin and Musashi in PSC-MSCs (ES-MSCs, iPS-MSCs-1, and iPS-MSCs-2) and BM-MSCs was analyzed by real-time PCR (qPCR). ***p < 0.005; compared to BM-MSCs. (B) Phase-contrast microscopy of BM-MSCs and various PSC-MSCs after being cultured 48 hours in neural differentiation medium (NDM; consist of RA (0.5 μM) in serum-free medium). Arrow indicates dendrite-like processes. (C) Gene expression of Nestin, Musashi, and MAP2 in PSC-MSCs and BM-MSCs after culturing in control complete medium (C) and NDM for 48 hours as analyzed by qPCR. *p < 0.05 and **p < 0.01, compared to Control. Error bars represent ± SEM of three independent experiments. Scale bar: 20 μm.

Figure 2. PSC-MSCs express early neural markes after RhoA kinase (ROCK) inhibition

(A) Immunofluorescent (IF) staining was performed to analyze for Nestin (green fluorescence) and MAP2 (red fluorescence) expression in PSC-MSCs after NDM or complete medium with ROCK inhibition using Y-27632 (Y). Hoechst 33342 was used to detect nuclei (blue). (B) Expression levels of Nestin (green fluorescence) and F-actin (red fluorescence) were analyzed by IF staining in PSC-MSCs after blebbistatin treatment. Scale bar: 20 μm.

Figure 3. ROCK inhibition in serum-free (SF) conditions induce further neural lineage commitment of PSC-MSCs

(A) Phase-contrast microscopy of PSC-MSC cell morphology after ROCK inhibition with Y-27632 in complete medium (Y), NDM, and Y-27632 in serum-free medium (SF+Y). (B) Nuclear size of PSC-MSCs after culturing in Y, NDM, and SF+Y as quantified by Image J software. Nuclei were be detected by Hoechst 33342 (blue fluorescence). *p < 0.05; **p < 0.01; ***p < 0.005; N.S., not significant. (C) Gene expression of MAP2 in PSC-MSCs after culturing in Y, NDM, and SF+Y as quantified as analyzed by qPCR. All results are shown as mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; compared to control. Scale bar: 20 μm.

Figure 4. PSC-MSCs express committed neural lineage proteins after ROCK inhibition in SF conditions

Protein expression of (A) Nestin (green fluorescence) and β-III-tubulin (red fluorescence), or (B) Nestin (green fluorescence) and Doublecortin (red fluorescence) in PSC-MSCs after culturing in Y, NDM, and SF+Y as analyzed by IF staining. Nuclei were be detected by Hoechst 33342 (blue fluorescence). Scale bar: 20 μm.

Figure 5. PSC-MSCs have differentiation capacity of neural lineage more than BM-MSCs

(A) IF staining for protein expression of NeuN (red fluorescence) PSC-MSCs cultured in SF+Y. Nuclei were be detected by Hoechst 33342 (blue fluorescence). Scale bar: 20 μm. (B) PSC-MSCs express higher baseline levels of NSC genes Nestin and Musashi than BM-MSCs. Culturing of PSC-MSCs in NDM or with inhibition of ROCK-myosin II axis leads to neural lineage commitment. When ROCK inhibition of PSC-MSCs is performed under SF conditions, these progenitors can further differentiate into more committed neural cells.