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Cell Biology International (2007) 31, 1428–1435 (Printed in Great Britain)
Signals in pathological CNS extracts of ALS mice promote hMSCs neurogenic differentiation in vitro
Cui‑Ping Zhaoa, Cheng Zhangab*, Yi‑Hua Wangc, Sheng‑Nian Zhoud, Chang Zhoua, Wan‑Yi Lib and Mei‑Juan Yub
aDepartment of Neurology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, PR China
bStem Cells and Tissue Engineering Research Center of Sun Yat-sen University, Guangzhou 510080, PR China
cDepartment of Neurosurgery, Affiliated Hospital, Yang Zhou University, Yangzhou 225000, PR China
dDepartment of Neurology, Qi-Lu Hospital, ShanDong University, Jinan 250012, PR China


The capability of MSCs to differentiate into neurons has been proven by many studies. Recently, other studies have cast doubt on MSCs neurogenic differentiation with non-physiological chemical inducing agents in vitro. This present study was designed to use conditioned medium to investigate whether signals from pathological condition of ALS were competent to induce a program of neurogenic differentiation in expanded cultures of hMSCs. Incubation of hMSCs with conditioned medium prepared from CNS extracts of ALS mice (SOD1-G93A ALS mice) resulted in a time-dependent morphological change from fibroblast-like into neuron-like, concomitant with increase in the expression of Nestin and subsequent β-tubulin III, NSE and GAP43. Moreover, signals in pathological CNS extracts of ALS mice were more effective in promoting hMSCs neurogenic differentiation than those in physiological extracts of normal adult mice. These results show that pathological condition of ALS is endowed with capacity to induce hMSCs neurogenic differentiation and hMSCs have shown a potential candidate in cellular therapy for ALS.

Keywords: Differentiation, Mesenchyme, Stem cells, Amyotrophic lateral sclerosis.

*Corresponding author. Department of Neurology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, PR China. Fax: +86 020 87333122.

1 Introduction

Mesenchymal stem cells (MSCs) derived from marrow stroma give rise to not only mesenchymal cells but also cells from neuro-ectodermal lineage such as neurons, astrocytes and oligodendrocytes (Black and Woodbury, 2001; Mezey et al., 2000). MSCs are easily isolated from bone marrow, expanded in number within a short time and can be used for autologous transplantation. MSCs have been intracerebrally transplanted into different animal models of central nervous system (CNS) disorders with positive functional effects, including stroke (Li et al., 2002), spinal cord injury (Chopp et al., 2000) and Parkinson's disease (Li et al., 2001). Transplanted MSCs are detected immunoreactive for neuro-glia cell markers. But some studies reported the failure of transplanted bone marrow cells to differentiate into neural cells (Wehner et al., 2003; Castro et al., 2002). Most transplanted MSCs were found near or in blood vessels and preserved their identity as hematopoietic stem cells in the brain (Vallieres and Sawchenk, 2003). These apparently contradictory experimental results have raised important issues concerning the nature of MSCs plasticity.

In in vitro studies on differentiating MSCs into neuron-like cells, non-physiological chemicals were often used such as β-mercaptoethanol plus butylated hydroxyanisole (Woodbury et al., 2000), isobutylmethylxanthine plus dibutyryl cMAP (Deng et al., 2001) or 5-Aza-C plus growth factors (Kohyama et al., 2001). Paolo's (Bertani et al., 2005) study supported the notion that such morphological changes observed in MSCs were caused by a cytotoxic effect of BME, BHA and/or DMSO because high frequency of apoptosis presented. These inducing conditions were apparently different from host microenvironment of brain which MSCs were grafted into. It is necessary to assess survival, proliferation and differentiation of MSCs in pathological condition in vitro before grafting.

Stem cells therapy is being actively and enthusiastically considered for many intractable CNS disorders. ALS should be a good candidate of MSCs transplantation because there is no efficient therapy for it now. But it has been proven that neurons produce a large amount of oxidative radicals and microglia are greatly activated with a lot of inflammatory factors released in ALS. CSF from ALS patients could induce neuron and astrocyte cytotoxicity involving free radicals and peroxynitrite (Pehar et al., 2002; Terro et al., 1996). These factors are naturally suspected to damage normal cells. Are MSCs adapted to the pathological condition of ALS? Are signals from pathological condition of CNS disease ALS (amyotrophic lateral sclerosis) competent to induce a program of neurogenic differentiation in expanded cultures of hMSCs? In the present study, conditioned medium with CNS extracts of ALS mice (SOD1-G93A ALS mice) was used to investigate the effect of pathological condition on hMSCs. We focused on the fate, survival capacity of MSCs in this conditioned medium.

2 Materials and methods

2.1 Animals

Transgenic male mice B6SJL-TgN(SOD1-G93A)1GUR (No. 002726) were purchased from the Jackson Laboratory (Bar Harbour, ME), which overexpressed human SOD1 and carried the Gly93Ala mutation (Gurney et al., 1994). The colony was derived from them and was maintained by breeding male transgenic mice to naïve (B6×SJL/J)F1 dams. Offspring were genotyped for the SOD1-G93A transgene using PCR of DNA extracted from the blood of tail veins as outlined by Jackson laboratories. All animal experiments were performed according to institutional guidelines which were in compliance with national and international law and policies.

2.2 Preparation of human mesenchymal stem cells (hMSCs)

Heparinized bone marrow was obtained from iliac crest aspirates from healthy volunteer with an informed consent. Mononuclear cells were separated by centrifugation in a Ficoll–Hypaque gradient (1.077g/ml, Sigma, SL, USA) and suspended in Dulbecco's modified Eagle medium with low glucose content (L-DMEM, Invitrogen, CA, USA) containing 10% fetal bovine serum (FBS). Finally, the mononuclear cells were seeded at 1×106cells/cm2 in T-25cm2 culture flasks. Cultures were maintained at 37°C in a humidified atmosphere of air containing 5% carbon dioxide.

The culture medium was replaced every 3 days and non-adherent cells were discarded. When the culture flasks became 80–90% confluent, the adherent cells were detached with 2.5g/l trypsin in 1.0mM sodium ethylenediaminetetraacetic acid (Na2-EDTA; Invitrogen, CA, USA). The hMSCs were reseeded at 1:3 dilution in T-25cm2 culture flasks. The above manipulation was repeated up to the fifth passage.

2.3 Analysis of hMSCs by FCM

Flow cytometry (FCM) was used to detect the cell surface markers specific for hMSCs. The hMSCs of fifth passage were harvested with 0.25% trypsin and resuspended in PBS at 2×104cells per reaction tube. The hMSCs were fixed in 4% cold paraformaldehyde for 30min and washed with PBS containing 2% FBS. Cells were incubated with mouse anti-human CD29 (1:1000, Santa Cruz, CA, USA), anti-CD34 (1:1000, Santa Cruz, CA, USA), anti-CD44 (1:1000, Santa Cruz, CA, USA), anti-CD45 (1:1000, Santa Cruz, CA, USA) antibodies and FITC-labeled goat anti-mouse secondary antibodies (1:100, Santa Cruz, CA, USA). The samples were characterized by FCM.

2.4 hMSCs cultures in conditioned medium

hMSCs were cultured in conditioned medium in vitro with ALS CNS extracts. Brains and spinal cords of SOD1-G93A ALS mice at symptomatic stage (about 20 weeks of age) and normal (SOD1-G93A negative) mice were removed, placed on ice and the wet weight in grams was rapidly measured. The tissue pieces were homogenized by adding L-DMEM (40mg/ml L-DMEM) and were incubated on ice for 10min. The homogenate was centrifuged for 10min at 10,000g at 4°C. The supernatant was collected, passed through a filter of 0.22μm and stored at −80°C for treatment of hMSCs (Chen et al., 2002). The hMSCs of fifth passage were seeded in six-well plates at 1×106cells/cm2 and maintained with L-DMEM containing 10% FBS for 2 days prior to stimulation with tissue extracts. The medium was substituted with ALS mice CNS extracts for ALS-conditioned group (1000μg/ml of protein), normal mice CNS extracts for normal-conditioned group (1000μg/ml of protein), and DMEM containing only 0.5% FBS for the control group. After treatment, cells were examined in a phase-contrast light microscope (Olympus IX71).

2.5 MTT assay

After reaching 80% confluency, the cells of fifth passage were trypsinized and cultured in 96-well plates at 1×104/ml in L-DMEM containing 10% FBS with 0.1ml per well. After 2 days incubation, the cells were divided into four groups: hMSCs in L-DMEM containing 10% FBS; DMEM group in L-DMEM containing 0.5% FBS; ALS-conditioned group and normal-conditioned group. Six wells were used for every group at each time-point. After incubating cells for 1–4 days, 10μl of 5mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, SL, USA) in PBS was added to each well and the plate was incubated at 37°C for 3h. The medium was removed and 100μl of dimethyl sulfoxide (DMSO, Sigma, SL, USA) per dish was added. After agitation at 37°C for 10min, optical density for four groups was assayed at 490nm with an enzyme-linked immunosorbent assay plate reader (Bio-TEK Instruments). Test treatments were expressed as percentage of the hMSCs in L-DMEM containing 10% FBS±standard deviation (SD).

2.6 Cell viability analysis

To investigate whether pathological condition would damage hMSCs in vitro, apoptosis and death of hMSCs in conditioned medium were detected with FCM. After exposure to conditioned medium for 3 days, cells were harvested with 0.25% trypsin, rinsed twice and fixed with 1ml of 75% cold ethanol at 4°C overnight. Cell pellets were incubated with 10μg/ml RNAse and stained with 50μg/ml propidium iodide (PI) for 30min in the dark. Samples were analyzed using an FCM with an excitation wavelength of 488nm. The resulting histograms were analyzed by the program WinMDI29.

2.7 Immunofluorescence analysis

After exposure to ALS-conditioned and normal-conditioned medium for 1 and 2 days, cells were fixed with 4% paraformaldehyde in PBS for 20min and treated with 0.3% Triton X-100 for 30min at room temperature. After rinsing with PBS twice, cells were treated with 5% FBS for 1h at room temperature. Later, cells were incubated overnight at 4°C in antibody-containing blocking solution. The following primary antibodies were used: anti-Nestin (1:50, Santa Cruz, CA, USA), β-tubulin III (TuJ1, 1:100, Chemicon, CA, USA), NSE (1:200, Neomarkers, CA, USA), GAP43 (1:200, Sigma, SL, USA) and GFAP (1:50, GeneTech, ShangHai, China) antibodies. The cells were incubated with secondary antibody Cy3 labeled goat anti-mouse (1:300, Santa Cruz, CA, USA) or goat anti-rabbit (1:200, Santa Cruz, CA, USA) for 2h at room temperature. After rinsing them in PBS, the cells were counterstained with DAPI and examined under fluorescent microscope (Olympus IX71). For quantification, the numbers of immunoreactive cells were determined by counting specifically stained cells in at least six random fields per well in six-well plates. Experiments were done in triplicate.

2.8 RT-PCR assay

RT-PCR was performed on mRNA isolated from cells after exposure to conditioned medium. For the synthesis of cDNA, 200ng mRNA from each sample was resuspended in a 20μl final volume of the reaction mixture. For each set of primers for PCR, a dilution of cDNA was amplified for 20, 23, 25, 28, 30, 33, 35, 38, and 40 cycles to define optimal conditions for linearity and to permit semi-quantitative analysis of signal strength.

The primers:




β-tubulin III rev: GTA TCC CCG AAA ATA TAA ACA CAA A (315bps);




NSE rev: CCG ATC TGG TTG ACC TTG AGC A (254bps); and



PCR conditions were 95°C, 5min, followed by 95°C, 30s, and annealing for 30s; then 72°C, 45s. Annealing temperature and optimal total reaction cycles were GAPDH 56°C for 25 cycles, β-tubulin III 54°C for 30 cycles, NSE 56°C for 30 cycles, GFAP 58°C for 40 cycles and GAP43 56°C for 40 cycles. GAPDH served as an internal standard in all samples. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide (EB, Sigma, SL, USA). The electrophoresis photograph was analyzed with AlphaImager2200.

2.9 Western blot analysis

To investigate protein expression, &007E;35μg protein of conditioned cells in each sample at 2 days were separated on 12% acrylamide gel and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane. The blot was probed by an anti-NSE (1:500, Neomarkers, CA, USA), anti-GAP43 antibody (1:1000, Sigma, SL, USA), anti-GAPDH (1:10,000, Santa Cruz, CA, USA) and a horse radish peroxidase (HRP)-conjugated secondary antibody, then developed with enhanced chemiluminescence reagents (Pierce).

2.10 Statistical analysis

Data were presented as mean±SEM. The immunoreactive cells and MTT analysis were analyzed by Student's t test and two-ways ANOVA (analysis of variance) followed by a Newman–Keuls' post hoc analysis for multiple comparisons.

3 Results

After a serial passage of adherent cells, a homogeneous population of cells was attained (Fig. 1A). hMSC expressed CD29 (98.8%) and CD44 (99.7%), but not CD34 (a hematopoietic stem cell marker) or CD45 (leukocyte common antigen), indicating that these cells were of mesenchymal origin with high purity.

Fig. 1

Morphology and FCM analysis of hMSCs in the fifth passage: (A) phase-contrast images of the fibroblast-like morphology of hMSCs (200× magnification); (C–F) FCM analysis. hMSCs expressed CD29, CD44 (C, E), but not CD34 or CD45 (D, F), (B) was control.

After exposed to ALS and normal-conditioned medium, hMSCs progressively assumed a time-dependent morphology change from fibroblast-like to multipolar neuron-like cells as indicated in Figs. 2 and 4A and B. The region around the nucleus became narrow at 5h and a part of cytoplasm was elongated to give rise to one or more cellular processes from 12 to 24h. Morphological changes further evolved, and more cells presented a clear polarization of cytoplasm and chromatin of nucleus condensed with one or two larger nucleoli at 48h (Fig. 2B and D). The nucleoli and cytoplasm were intensely stained blue with cresyl violet at 48h (Fig. 2D). After 3 days, a network-like structure among cells came into being (Fig. 2C). None of these changes were observed in control group (DMEM containing 0.5%FBS; see Fig. 2A).

Fig. 2

Time-dependent morphology change of hMSCs in ALS-conditioned medium and immunoreactivity to neurogenic markers. (A) Phase-contrast of hMSCs in DMEM containing 0.5% FBS (DMEM in abbreviation) at 2 days. hMSCs remained fibroblast-like morphology; (B) phase-contrast of hMSCs in ALS-conditioned medium (A condition in abbreviation) at 2 days. The region around the nucleus became narrow and a part of cytoplasm was elongated to give rise to one or more cellular processes (arrow); (C) phase-contrast of hMSCs in ALS-conditioned medium at 3 days and a network-like structure among cells came into being; (D) staining with cresyl violet of hMSCs in ALS-conditioned medium at 2 days. The cells presented a clear polarization of cytoplasm, chromatin of nucleus condensed with one or two larger nucleoli (arrow) (A–D, bar=100μm); (E–J) immunostaining of hMSCs in DMEM containing 0.5% FBS and ALS-conditioned medium. Nestin expression in DMEM (E) and in ALS-conditioned medium (F) at 12h; β-tubulin III expression in ALS-conditioned medium (H) at 1 day but not in DMEM (G); NSE expression in ALS-conditioned medium (J) at 2 days but not in DMEM (I). (E, F, H, J) Bar=100μm. (G, I): 200× magnification. (Note: “A condition” was the abbreviation for ALS-conditioned medium, and “DMEM” was abbreviation for DMEM containing 0.5% FBS.)

In further analysis, hMSCs in both kinds of conditioned medium were detected to express immature and mature neuron markers Nestin, β-tubulin III, NSE and GAP43. Immunofluorescence analysis showed that at first 12h the expression of Nestin increased and followed with a decrease at 24h in ALS-conditioned medium (Fig. 2F). Less than 1% of hMSCs were positive to Nestin in control (Fig. 2E). The early neuron marker β-tubulin III was expressed by 36.5% of cells at 1 day in ALS-conditioned medium (Fig. 2H), and peaked at 2 days with up to 48.7% of cells immunopositive, while there were 41.2% of cells NSE positive (t=2.65, P=0.013<0.05) (Fig. 4A and B) in normal-conditioned medium. hMSCs in control were not positive for β-tubulin III and NSE (Fig. 2G and I). There was no cell positive to GFAP at any time-point observed in both conditioned medium or in control.

In semi-quantitative RT-PCR analysis, relative abundance of β-tubulin III transcripts increased in both kinds of conditioned medium and maximized at 1 day; mRNA of NSE increased with time in 2 days. GAP43 was weakly expressed at 2 days but significantly increased compared to that prior to exposure to conditioned medium (Fig. 4C and E).

By semi-quantitative analysis with RT-PCR and Western blot, relative abundance of NSE and GAP43 expression in ALS-conditioned medium at 2 days was found higher than those in normal-conditioned medium (NSE mRNA: t=8.731, P<0.01; GAP43 mRNA: t=3.828, P<0.05, Fig. 4C and E. NSE protein: t=92.507, P<0.01; GAP43 protein: t=28.747, P<0.01, Fig. 4D and F). These data indicated that there existed stronger signals in pathological condition of ALS than in physiological condition of adult mice to promote hMSCs differentiation into neuron-like cells.

MTT assay was performed to investigate the effect of conditioned medium on the growth and survival of hMSCs. As indicated in Fig. 3A, cells in both kinds of conditioned medium grew gradually and the proliferation was delayed compared to in complete growth medium (DMEM containing 10% FBS). The values at 3 days were 71.3% in ALS-conditioned medium and 82.7% in normal-conditioned medium (P<0.01 and P<0.05, respectively), only 62.6% and 66.3%, respectively at 4 days compared to in complete growth medium (P<0.01 and P<0.01, respectively). The value in normal-conditioned medium seemed to be higher than that in ALS-conditioned but there was no significant difference (P>0.05). In cell viability analysis with PI staining by FCM, there were no detectable apoptosis and death in both kinds of conditioned medium (Fig. 3B and C) at 3 days when the growth began delayed. It suggested that the delayed proliferation might result from neuron-like differentiation of cells not from cell death.

Fig. 3

Cell survival and viability. (A) MTT assay: the growth was delayed in both kinds of conditioned medium compared to in complete growth medium (DMEM containing 10% FBS; *P<0.01, #P<0.05). (B) There were no detectable apoptosis and death in ALS-conditioned medium (B) or in normal-conditioned medium (C) at 3 days. (Note: “A condition” was the abbreviation for ALS-conditioned medium, and “N condition” was the abbreviation for normal-conditioned medium.)

Fig. 4

Neurogenic differentiation of hMSCs in both conditioned medium. Cells (48.7%) became NSE positive in ALS-conditioned medium (A) while there were 41.2% of cells NSE positive at 2 days (P<0.05) in normal-conditioned medium (B) (200× magnification) (Note: “A condition” was the abbreviation for ALS-conditioned medium, “N condition” was abbreviation for normal-conditioned medium.) mRNA transcripts of neurogenic markers (C) and relative expression (E). In (C) Lanes 1, 2, 3, 4 and 5 denoted 0 day, 1 day ALS-conditioned, 1 day normal-conditioned, 2 days ALS-conditioned and 2 days normal-conditioned, respectively. Western blot analysis of NSE and GAP43 (D) and relative expression (F). In (D) Lanes 1, 2 and 3 denoted 0 day, 2 days ALS-conditioned and 2 days normal-conditioned, respectively. GAPDH was internal control. *P<0.01, #P<0.05, compared to cells in normal-conditioned medium at the same time-point.

4 Discussion

Neurogenic differentiation of MSCs both in vitro and in vivo has been reported by numerous investigators (Sanchez-Ramos, 2002). However recently, other studies have cast doubt on MSCs neural differentiation with chemical inducing agents (Bertani et al., 2005; Neuhuber et al., 2004; Lu et al., 2004). The morphological change of cells and expression of neuron markers were rapid and transient after exposure to DMSO/BHA (Choi et al., 2006) with high percentage of apoptotic cells present (Rismanchi et al., 2003). So some studies focused on the search of physiological inducer including growth factors such as BDNF, NGF and EGF. But response of MSCs in pathological condition has not been studied, especially in that of neurodegenerative disease.

Our main findings were as follows: (A) a time-dependent morphological change in hMSCs from fibroblast-like to neuron-like was observed after incubation with ALS-conditioned medium; (B) concomitant with the morphological changes, the cells became positive for neuron markers β-tubulin III, NSE and GAP43; (C) hMSCs survived the pathological ALS-conditioned medium and underwent neurogenic differentiation without detectable apoptosis and death; and finally, (D) the changes in morphology, neurogenic immunophenotype were also elicited by normal CNS but neurogenic differentiation signals in pathological condition of ALS mice were more effective than those in normal adult mice.

It has been reported that microenvironment affects the fate of MSCs. In a co-culture system with postnatal hippocampal slice, MSCs displayed neuron morphology and expressed the neuron marker NeuN (Abouelfetouh et al., 2004). Another study found that MSCs were induced a neuron-like phenotype and a significant increase on the expansion in vitro by adult hippocampus derived soluble factors (Rivera et al., 2006). Co-cultured with 4% paraformaldehyde-fixed cerebellar granule neurons, as described recently by Wislet-Gendebien et al. (2005), hMSCs were induced to form neuronal elements in vitro. Our experimental protocol involved conditioned medium with CNS extracts of ALS mice to emulate the pathological condition in vivo. Although exact molecular mechanism was unraveled, our findings strongly suggested that hMSCs were sensitive to CNS of ALS and underwent the neurogenic differentiation process. These findings were confirmed by morphology changes, neurogenic immunophenotype, mRNA expression and Western blot. However we only called them neuron-like cells because we did not verify the functional electrical activity of the cells.

Our work has shown that the pathological condition in ALS mice was more effective in promoting hMSCs neurogenic differentiation than normal adult CNS. This result is consistent with transplantation studies in vivo indicating that the intact adult brain has less capacity to direct the stem cells differentiation than the developing brain and injured brain (Munoz-Elias et al., 2004; Coyne et al., 2006; Bjorklund and Lindvall, 2000). hMSCs conditioned by traumatic brain extracts in vitro were found a time-dependent secreting many growth factors including BDNF, NGF, bFGF, VEGF and HGF. Moreover the production of growth factors conditioned by traumatic brain extracts was more than conditioned by normal brain extracts (Chen et al., 2002). We hypothesize that hMSCs response to ALS CNS extracts and produce growth factors which elicit the neurogenic process. In ALS, microglia and astrocytes are activated with large amount of cytokines, chemokines released including IL1α, IL1β, IL1RA, IL2, IL3, IL4, TNFα, TGFβ and glutamate. We do not exclude the possibility that these factors are present and involved in the neurogenic effects on hMSCs.

It was beyond our preconception that hMSCs survived the pathological ALS-conditioned medium without detectable apoptosis and death. This result suggested hMSCs were not prone to be damaged under this condition. hMSCs showed a delayed growth in conditioned medium which was most likely due to the differentiation not due to death. The observation that undifferentiated hMSCs also expressed immature and mature neuron markers confirmed the data of other groups (Bertani et al., 2005; Choi et al., 2006; Blondheim et al., 2006; Kim et al., 2005; Bossolascoa et al., 2005). MSCs seemed to already contain a subpopulation of cells capable of neural differentiation or a neural predisposition.

Although a comprehensive analysis of the component(s) responsible for the neurogenic effects has not been performed, our results show that under ex vivo conditions, MSCs differentiation to neurogenic lineage is feasible under the influence of pathological condition in ALS. It appears interesting to speculate that the implant of ex vivo expanded and manipulated MSCs into a pathological CNS milieu may represent a potential therapy for CNS disorder. This proposed protocol will be more feasible than transplantation of neural stem cells. In summary, data presented here show that the pathological condition of CNS provides sufficient signals to promote hMSCs neurogenic differentiation in vitro. The identification and characterization of the molecular mechanism will have significance not only for the understanding of MSCs biology but also for the field of neurogenesis and stem cell transplantation research.


This work is supported by grants from the CMB Fund (4209347) and the Key Project of the State Ministry of Public Health (2001321).


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Received 9 January 2007/13 May 2007; accepted 6 June 2007


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