In the present study, we thoroughly assessed for the first time the capacity of CART to enhance the neural differentiation of MSCs. We demonstrated that, when exposed to CART, MAP-2, GFAP or NeuN positive cells were traced in the cultured MSCs. Furthermore, the proposed differentiated neurons not only have the potency to express ChAT and TH, but also display the Nissl bodies in the cytoplasm. In addition, neurotrophic factors including NGF and BDNF were up-regulated in CART-treated group. Our results suggest that CART may promote the differentiation of MSCs in vitro by enhancing the endogenous expression of NGF and BDNF.
MSCs extracted from mouse bone marrow have great multiplication potency. Cell-doubling time is 48-72 h and cells can be expanded in culture for more than 60 doublings . Herein, we isolated BM-MSCs with the characteristics involving adherence to cell culture plastic, specific surface antigen expression, and multipotent differentiation which met the criterion defined by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy . Also, BM-MSCs possess pluripotency defined by their ability to differentiate into bone, fat, cartilage and muscle [1, 12–14]. Compelling evidences unveiled that MSCs are competent to break germ layer commitment and differentiate into cells expressing neuroectodermal properties . Such a capacity opens extensive possibilities for autologous therapeutic treatments in a variety of neurological disorders. Also, as there are ethical issues related to isolating stem cells from fetal tissue and from adults, alternative sources of neuron-like cells for cell therapy are necessary. Indeed, a portion of studies indicate that, in contrast to the primary stem cells, the transplantation of pre-differentiated stem cells can diminish the risk of tumogenesis in the host patients . However, current inducing methods obtain a lower percentage of neuronal differentiation by a long inducing time . In 2000, Sanchez-Ramos et al used RA in combination with BDNF to introduce differentiation of BMSC to neural phenotypes. NeuN and GFAP positive cells only rank 0.5 and 1% respectively . Likewise, Woodbury et al found β-mercaptoethanol (BME) can rapidly induce the transition from MSCs into neurons but not glial cells . There is also a debate about whether or not the converted neural phenotyped cells are able to work . This premise provided the impetus to investigate better and new reagent which can yield functional neuronal cells at a high rate.
Here we extended our study on the neuroectodermal conversion of BM-MSCs in vitro by exposure to the prospective peptide CART. After treatment with CART, we found that CART could successfully differentiate MSCs into neural cells in vitro. These cells were morphologically similar to neural stem cells. More importantly, they could express neural progenitor stem cell marker Nestin and mature glial marker GFAP as well as mature neuron markers like MAP-2 and NeuN. It indicated that the protocol of using CART for three or six days induced significant changes in morphology and expression of markers of early and mature neural cell types. Similar with induction rate of bFGF plus EGF, CART exposure obtained that MAP-2, GFAP and NeuN expressions were found in 30.8 ± 4.7%, 20.5 ± 2.5%, 32.1 ± 2.3% of all cells in 3 days, and 41.2 ± 3.1%, 21.3 ± 2.2%, 40.3 ± 2.7% in 6 days (Figure 4). However, the expression of one or even two neuronal proteins does not prove that the cell bearing these "neuronal markers" is capable of all the complex functions of a neuron. It will be important to determine whether these cells possess functional characteristics of neurons. Subsequently, double-labeled immunofluorescence was performed to calculate the function of the converted cells. It was intriguing that the differentiated NeuN positive cells co-expressed ChAT or TH. Our study on excitable property of MSC-derived neural like cells demonstrated that the differentiated cells pocess founctional patassium and cacium channels (Additional file 1). Recent research discovered that the neuron-like cells differentiated from BM-MSCs resemble the endogenous neural progenitors in morphological, immunocytochemical, and functional characteristics . Another independent investigator also reported that the transformed BM-MSCs exhibit both neuron-like biochemical function and some corresponding electrophysiological properties .
CART is an endogenous neuropeptide which is widely distributed in the brain and peripheral nervous system. It has been well-established that CART can promote the survival and differentiation of neurons. Some investigators disclosed that CART has the capability to modulate the expression of growth factors and neurotrophic factors, which probably participated in the underlying effect of CART . Based on this theory, we made an assumption that neurotrophic factors could be the bridge between CART and stem cell differentiation. In order to verify this hypothesis, the amount of NGF and BDNF was determined by RT-PCR. The result that neurotrophic factors are significant higher in CART treated group implied CART could promote the endogenous production of neurotrophic factors. Neurotrophic factors in cell and tissue culture have been shown to promote neuronal survival and differentiation and also regulate the growth of axons and dendrites [24, 25]. The presence of growth factors such as BDNF or NGF in the transplantation or the co-culture experiment are considered as physiological inductors for neural differentiation of MSCs [8, 9]. MSCs can induce a variety of neuro-regulatory proteins in addition to BDNF and β-NGF . Further study demonstrated that the NGF and BDNF receptor TrkA and TrkB do exist on the surface of MSCs . Moreover, the TrkB receptor modified by BDNF may trigger the MAPK signal pathway. This process contributes to the maturation and neural differentiation of MSCs . Early studies confirmed that BDNF+RA (retinoic acid) adult bone marrow stromal cells can be induced to differentiate into neural cells in vitro . However, CART stimulated the endogenous BDNF and NGF expression of MSCs. This stimulation may imitate the natural physiological pattern. As a result, we obtained a higher yield rate than in previous studies. In the hippocampus, BDNF and other neurotrophins are expressed from early stage of neuron development [29, 30]. This process is a prerequisite for the differentiation of neurons derived from hippocampal precursor cells in vitro [31–33]. Overall, findings suggest that the BDNF and NGF enhanced by CART may be the foundation of stem cell differentiation and maturation.