Neonatal brain damage caused by hypoxia and anemia before birth is known to be one important cause of infant death at birth and long-term neurological deficits, such as cerebral laceration and mental retardation [1-5]. However, as treatment strategies for neonatal encephalopathy are still rare, ongoing research remains important. It has previously been shown that mesenchymal stem cells (MSC) are able to improve the repair of various disease tissues, ranging from cardiovascular to graft-versus-host diseases [6,7]. MSC are used as a valuable therapeutic tool because of the lack of expression of MHCII, costimulatory proteins (CD80, CD86 and CD40) and immunogenicity [8-10]. Therefore, several clinical trials have focused on studying the effectiveness and safety of using MSC for a variety of pathological treatment options .
However, current conventional in vitro culture methods are often not able to maintain the stability of MSC, which manifests as aging and osteoblast autonomous differentiation during MSC the culturing process [12-15]. This causes cell volume to increase and proliferation decrease, which seriously affects its aforementioned roles. Autonomous and premature differentiation of MSC reduces their ability to form other cell lineages, such as neuron cells and cardiomyocytes. In addition, during the MSC in vitro amplification process, the issues of aging and autonomous osteogenic differentiation seriously affect the therapeutic effectiveness and safety of treatments with MSC.
The critical factor in maintaining the original characteristics of MSC is based on culture conditions. MSC cultured in a stereoscopic environment are less likely to differentiate, and the hanging drop method of culturing keeps MSC in an undifferentiated state and maintains their high migration properties. In this study, we compared the therapeutic effectiveness of MSCs cultured by hanging drop and conventional culturing methods on the treatment of brain injured mice.
2 Materials and Methods
2.1 MSCs cell culture
The frozen first generation bone marrow-derived hMSCs were provided by Shenzhen Baiwang Biological Technology Co., Ltd. Twenty-four (24) hours after resuscitation, hMSCs reached low density (100 cells / cm2) and then were cultured in complete culture medium (CCM) containing 17% FBS for 7-8 days until about 70% confluent. hMSCs were passaged under the same conditions, but those used for the experiments were only of the first three generations. Each drop of complete culture medium containing 10 000-250 000 cells on a glass slide were hanging drop cultured for four days. In order to obtain spheroid derived cells, the spheroids were incubated using trypsin / EDTA for 5 – 30 min depending on the size of spheroids, pipetted every 2-3min to disperse the cells, and their sizes observed under the microscope. The second group of MSCs were conventionally in polyethylene culture plates.
2.2 Preparation of the brain injured mouse models and MSCs treatment
Nine-day-old C57BL/6 mice (Experimental Animal Center of Henan Province) were each subjected to hypoxic-ischemic (HI) brain injury as follows: anesthetized with isoflurane, hypoxia at 10% oxygen for 45 minutes, and permanent occlusion at the right carotid artery. The control group mice were only subjected to arteriotomy after anesthetization. The HI process led to a 10% death rate. The surviving HI mice were randomly divided into three groups with 5 mice each. The first treatment group was administered 1 × 106 MSC derived via hanging culture, the second treatment group 1 × 106 MSC derived conventionally, and the control group a saline solution. All administrations were performed by nasal aspiration. PBS containing 3ml of hyaluronidase were dropped inside both of the nostrils to increase the permeability of the mucosa of mice, againafter 30 minutes. In addition, igloos and walking rounds were placed in the baskets to support physical exercise induce nerve regeneration [16,17]. The mouse brains were analyzed using MRI after 28 days.
2.3 RNA extraction and real time RT-qPCR analysis
Total RNA was extracted using Trizol reagent (Invitrogen, COUNTRY), according to operating reference reagent instructions. cDNA synthesis and PCR amplification were conducted with a RevertAid First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany). RT-qPCR was performed using a DyNAmo Flash SYBR Green qPCR kit (Finnzymes Oy, Espoo, Finland). The expression levels were calculated and normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
2.4 Statistical analysis
Data are expressed as mean ± S.E.M. The statistical evaluation of the data was carried out using ANOVA and Pearson’s correlation test were analyzed by SPSS 12.0.1. P < 0.05 was considered statistically significant. The ratio of MSCs mRNA expression level to GADPH mRNA expression level cultured by conventional method was defined as 1.
3.1 Morphology observation of hanging drop cultured MSCs
Conventionally cultured MSCs showed typical long cablelike morphology after 7 days. The hanging drop cells on the other hand showed a spheroid morphology after 2 days. After digestion with trypsin, hanging drop MSC were one fourth the diameter of the conventional cells.
3.2 Morphology impact of spheroidal formation on the expression of sternness marker genes
To understand regulation effect of the MSCs stemness marker genes during the culturing process, expression levels of Oct4, Sox2, Nanog, and the migration-related gene CXCR4 mRNA, in the basic media were analyzed. Real time RT-PCR analysis (Figure 1) showed that the expression levels of all four genes significantly (P < 0.05) increased, compared to those of the conventional group. This indicates that the formation of spheroidal MSCs obtained from the hanging drop culture could help maintaining the expression levels of the MSCs stemness genes and migration gene.
3.3 Treatment of HI mouse models
MSCs was intranasally aspirated to the HI mice. The brain injury healing conditions of different mice groups were analyzed by MRI on the 28th day following treatment. Hanging drop cultured MSCs better healed the HI mice brain injury than those cultured by the conventional method, and both of these treatment groups healed better than those not injected with MSCs, which were consistent as expected (Figure 2). This demonstrates that the MSCs have certain therapeutic effect on brain injury caused by HI.
Brain damage caused by hypoxia still have no ideal treatment. A number of studies [18-22] have shown that mesenchymal stem cells have migration capability and can differentiate into nerve cells at the site of injury. In addition, MSCs can provide cytokines and growth factor in the paracrine manner to improve tissue regeneration. However, the conventional MSCs culturing method often causes MSCs differentiation, resulting in weakened stemness and diminished migration ability. MSCs migration ability is an important characteristic of the MSCs, which is not only the foundation of MSCs’ autonomous migration to the damaged tissue after intravenous injection, but also the theoretical basis of MSCs intravenous injection treatment of visceral injuries and systemic diseases, such as myocardial infarction . Recent studies indicate that inflammatory chemokine receptors, especially CCR1 and CCR2, are closely related to MSCs migration ability in/during myocardial infarction [24-28]. The expression reduction of MSCs surface migration related receptor and oversize cause a decline or loss of MSCs migration capability, resulting in more than 70% of MSCs obstructed in the lung after intravenous injection and only a very small amount (<5%) of the cells reached the infarct cardiac tissue [29-32]. The low migration ability inevitably affected the targeting ability and effectiveness of the MSCs.
In this study, the hanging drop cultured MSCs and conventional cultured MSCs were used to treat HI mouse models. The MSCs obtained from the hanging drop culture were spheroids. Under the effects of trypsin / EDTA and using the ordinary optical microscope, the sizes of the hanging drop cultured MSCs were significantly smaller in diameter than those of the MSCs obtained from conventional culture (the former was about 1/3-1/4 of the later). Real time RT-qPCR was used to analyze the expression levels of sternness genes, Oct-4, Sox2 and Nanog and migration-related gene CXCR4 for the MSCs derived from the hanging drop culture and conventional culture. The results indicate that the MSCs obtained from hanging drop culture had higher expression levels of these genes than the MSCs obtained from conventional culture, strongly suggesting that the differentiation of hanging drop cultured MSCs was lower than the conventional cultured MSCs. In addition, the MSCs obtained from the hanging drop culture had higher expression levels of migration related genes CXCR4 than the MSCs obtained by the conventional culture. These results demonstrated that the MSCs cultured by the hanging drop method had relatively low differentiation and higher migration ability. In the treatment experiments of the HI models, the MRI results showed that the MSCs obtained from the hanging drop culture were more effective at repairing brain damage resulting from HI. In conclusion, hanging drop cultured MSCs had higher expression levels in stemness genes and migration related genes CXCR4 than those of the conventional cultured MSCs. Intranasal injection of MSCs HI-damage in mice by differentiating into nerve cells, repairing nerve damage, reducing scar forming. This may provide a new therapeutic technique for brain injury treatment in mice and other species.
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About the article
Published Online: 2016-12-26
Published in Print: 2016-01-01
Conflict of interest: Authors declare nothing to disclose.
Citation Information: Open Life Sciences, Volume 11, Issue 1, Pages 519–523, ISSN (Online) 2391-5412, DOI: https://doi.org/10.1515/biol-2016-0068.
© 2016 Yongli Lou et al.. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0