Effectiveness of mesenchymal stems cells cultured by hanging drop vs. conventional culturing on the repair of hypoxic-ischemic-damaged mouse brains, measured by stemness gene expression

Yongli Lou; Dewei Guo; Hui Zhang; Laijun Song

doi:10.1515/biol-2016-0068

Artikel Open Access

Effectiveness of mesenchymal stems cells cultured by hanging drop vs. conventional culturing on the repair of hypoxic-ischemic-damaged mouse brains, measured by stemness gene expression

Yongli Lou , Dewei Guo , Hui Zhang und Laijun Song

Veröffentlicht/Copyright: 26. Dezember 2016

Veröffentlicht von

Veröffentlichen auch Sie bei De Gruyter Brill

Manuskript einreichen Informationen für Autor*innen Erkunden Sie dieses Fachgebiet

Aus der Zeitschrift Open Life Sciences Band 11 Heft 1

Abstract

In this study, we investigated the therapeutic effects of Human Mesenchymal Stem Cells (hMSCs) cultured by hanging drop and conventional culturing methods on cerebellar repair in hypoxic-ischemic (HI) brain injured mice. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to analyze the expression levels of three stemness genes, Oct4, Sox2 and Nanog, and the migration related gene CXCR4. MSC prepared by hanging drop or conventional techniques were administered intranasally to nine day old mice, and analyzed by MRI at day 28. Results indicate that the MSCs, especially the hanging drop cultured MSCs, significantly improved the mice’s cerebellar damage repair. MSCs derived from the hanging drop culture were smaller than those from the conventional culture. The gene expression levels were significantly increased for the MSCs derived from the hanging drop culture. The mechanism might relate to the fact that the hanging drop cultured MSCs can be kept in an undifferentiated state, resulting in its higher expression level of migration receptor of CXCR4.

Keywords: MSC; stemness; brain injury

1 Introduction

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 [11].

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 Results

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.

Figure 1

Real time RT-PCR analyses for stemness gene Oct-4, Sox2 and Nanog and migration-related gene CXCR4 in four groups.

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.

Figure 2

MRI analysis of HI mouse brain; A: no HI injury; B: untreated HI models; C: PBS treatment; D: Treatment of MSCs cultured by conventional method; E: Treatment of MSCs cultured by hanging drop method.

4 Discussion

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 [23]. 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.

Conflict of interest: Authors declare nothing to disclose.

References

[1] Dammann O., Ferriero D., Gressens P., Neonatal encephalopathy or hypoxic ischemic encephalopathy? Appropriate terminology matters, Pediatr. Res, 2011, 70, 1–2.10.1203/PDR.0b013e318223f38dSuche in Google Scholar

[2] De Haan M., Wyatt J. S., Roth S., Vargha-Khadem F., Gadian D., et al., Brain and cognitive-behavioural development after asphyxia at term birth, Dev. Sci., 2006, 9, 350–358.10.1111/j.1467-7687.2006.00499.xSuche in Google Scholar

[3] Ferriero D. M., Neonatal brain injury, N. Engl. J. Med., 2004, 351, 1985–1995.10.1056/NEJMra041996Suche in Google Scholar

[4] Graham E. M., Ruis K. A., Hartman A. L., Northington F. J., Fox H. E., A systematic review of the role of intrapartum hypoxiaischemia in the causation of neonatal encephalopathy, Am. J. Obstet. Gynecol., 2008, 199, 587–595.10.1016/j.ajog.2008.06.094Suche in Google Scholar

[5] Van Handel M., Swaab H., de Vries L. S., Jongmans M. J., Long-term cognitive and behavioural consequences of neonatal encephalopathy following perinatal asphyxia: a review, Eur. J. Pediatr., 2007, 166, 645–654.10.1007/s00431-007-0437-8Suche in Google Scholar

[6] Wang C. C., Chen C. H., Lin W. W., Hsieh P. C. H., Lai P. H., et al., Direct intramyocardial injection of mesenchymal stem cell sheet fragments improve cardiac functions after infarction, Cardiovasc. Res., 2008, 77, 515–524.10.1093/cvr/cvm046Suche in Google Scholar

[7] Gonzalo-Daganzo R., Regidor C., Martin-Donaire T., Rico M. A., Bautista G., et al., Results of a pilot study on the use of third -party donor mesenchymal stromal cells in cord blood transplantation in adults, Cytotherapy, 2009, 11, 278-288.10.1080/14653240902807018Suche in Google Scholar

[8] Deans R. J., Moseley A. B., Mesenchymal stem cells: Biology and potential clinical uses, Exp. Hematol., 2000, 28: 875-884.10.1016/S0301-472X(00)00482-3Suche in Google Scholar

[9] Shi M. X., Chen R., Guo C., Gao L., Influence of ketamine on amino acid neurotransmitters secretion by nerve cells in vitro, Bangladesh J. Pharmacol., 2016, 11, 55-60.10.3329/bjp.v11iS1.26411Suche in Google Scholar

[10] Zhang R., Liu Y., Yan K., Chen L., Chen X. R., et al., Anti-inflammatory and immunomodulatory mechanisms of mesenchymal stem cell transplantation in experimental traumatic brain injury, J Neuroinflammation, 2013, 10, 106–118.10.1186/1742-2094-10-106Suche in Google Scholar PubMed PubMed Central

[11] Salem H. K., Thiemermann C., Mesenchymal stromal cells: Current understanding and clinical status, Stem Cells, 2010, 28, 585–596.10.1002/stem.269Suche in Google Scholar PubMed PubMed Central

[12] Baxter M. A., Wynn R. F., Jowitt S. N., Wraith J. E., Fairbairn L. J., and Bellantuono I., Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion, Stem Cells, 2004, 22, 675-682.10.1634/stemcells.22-5-675Suche in Google Scholar PubMed

[13] Wen S. J., Song S. B., On the effect of health insurance on Chinese rural elders’ health demand, Acta Med. Mediterr., 2016, 32, 481-484.Suche in Google Scholar

[14] Krampera M., Pasini A., Rigo A., Scupoli M. T., Tecchio C., Malpeli G., Scarpa A., Dazzi F., Pizzolo G., Vinante F., HB-EGF/ HER-1 signaling in bone marrow mesenchymal stem cells: inducing cell expansion and reversibly preventing multilineage differentiation, Blood, 2005, 106, 59-66.10.1182/blood-2004-09-3645Suche in Google Scholar PubMed

[15] Wagner W., Horn P., Castoldi M., Diehlmann A., Bork S., Saffrich R., Benes V., Blake J., Pfister S., Eckstein V., et al., Replicative senescence of mesenchymal stem cells: a continuous and organized process, PLoS One, 2008, 3, e2213.10.1371/journal.pone.0002213Suche in Google Scholar PubMed PubMed Central

[16] Wang L. F., Tian X. Y., Wang B. C., Yin M. N., Effect of comprehensive nursing intervention on depression, anxiety and quality of life of infertility patients, Acta Med. Mediterr., 2016, 32, 515-518.Suche in Google Scholar

[17] Van Praag H., Kempermann G., Gage F. H., Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus, Nat. Neurosci., 1999, 2, 266–270.10.1038/6368Suche in Google Scholar PubMed

[18] Pearson Yanthe E.; Lund Amanda W., Lin, Alex W. H., Non-invasive single-cell biomechanical analysis using live-imaging datasets, J. Cell Sci., 2016, 129, 3351-3364.10.1242/jcs.191205Suche in Google Scholar PubMed

[19] Zong D., Yin L., Zhong Q., et al., ZNF488 Enhances the Invasion and Tumorigenesis in Nasopharyngeal Carcinoma Via the Wnt Signaling Pathway Involving Epithelial Mesenchymal Transition, Cancer Res. Treat., 2016, 48, 334-344.10.4143/crt.2014.311Suche in Google Scholar PubMed PubMed Central

[20] Fernanda F. C., Macedo R., Patricia R., Hypoxic preconditioning enhances mesenchymal stromal cell lung repair capacity, Stem Cell Res. Ther., 2015, 6. 120-123.10.1186/s13287-015-0120-3Suche in Google Scholar PubMed PubMed Central

[21] Lu M. Y., Jolly M. K., Onuchic J. Ben-Jacob E., Toward Decoding the Principles of Cancer Metastasis Circuits, Cancer Res., 2014, 74, 4574-4587.10.1158/0008-5472.CAN-13-3367Suche in Google Scholar PubMed

[22] Yang N., Hui L., Wang Y., Yang H. J., Jiang X. J., Overexpression of SOX2 promotes migration, invasion, and epithelialmesenchymal transition through the Wnt/beta-catenin pathway in laryngeal cancer Hep-2 cells, Tumor Biol., 2014, 35, 7965-7973.10.1007/s13277-014-2045-3Suche in Google Scholar PubMed

[23] Pittenger M. F., Martin B. J., Mesenchymal stem cells and their potential as cardiac therapeutics, Circ. Res., 2004, 95, 9-20.10.1161/01.RES.0000135902.99383.6fSuche in Google Scholar PubMed

[24] Shi G. Q., Yang J., Liu J., Liu S. N., Song H. X., Zhao W. E., Liu Y. Q., Isolation of flavonoids from onion skin and their effects on K562 cell viability, Bangladesh J. Pharmacol., 2016, 11, 18-25.10.3329/bjp.v11i0.26419Suche in Google Scholar

[25] Belema-Bedada F., Uchida S., Martire A., Kostin S., Braun T., Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2, Cell Stem Cell, 2008, 2, 566-575.10.1016/j.stem.2008.03.003Suche in Google Scholar PubMed

[26] Huang J., Zhang Z., Guo J., Ni A., Deb A., Zhang L., Mirotsou M., Pratt R.E., Dzau V.J., Genetic modification of mesenchymal stem cells overexpressing CCR1 increases cell viability, migration, engraftment, and capillary density in the injured myocardium, Circ. Res., 2010, 106, 1753-1762.10.1161/CIRCRESAHA.109.196030Suche in Google Scholar PubMed PubMed Central

[27] Miao M. S., Wang T., Li Y., Li M., Guo L., Zhang Y., Effect of Cynomorium flavonoids on mouse model of perimenopausal depression, Bangladesh J. Pharmacol., 2016, 11, 31-35.10.3329/bjp.v11iS1.25638Suche in Google Scholar

[28] Schenk S., Mal N., Finan A., Zhang M., Kiedrowski M., Popovic Z., McCarthy P.M., Penn, M. S., Monocyte chemotactic protein-3 is a myocardial mesenchymal stem cell homing factor, Stem Cells, 2007, 25, 245-251.10.1634/stemcells.2006-0293Suche in Google Scholar PubMed

[29] Gao Y. C., Effects of exercise on oxidative stress and related factors of obese young women, Acta Med. Mediterr., 2016, 32, 563-566.Suche in Google Scholar

[30] Assis A.C., Carvalho J.L., Jacoby B.A., Ferreira R.L., Castanheira P., Diniz S.O., Cardoso V.N., Goes A.M., Ferreira A.J., Time-dependent migration of systemically delivered bone marrow mesenchymal stem cells to the infarcted heart, Cell Transplant, 2010, 19, 219-230.10.3727/096368909X479677Suche in Google Scholar PubMed

[31] Barbash I.M., Chouraqui P., Baron J., Feinberg M.S., Etzion S., Tessone A., Miller L., Guetta E., Zipori D., Kedes L.H., et al., Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution, Circulation, 2003, 108, 863-868.10.1161/01.CIR.0000084828.50310.6ASuche in Google Scholar PubMed

[32] raitchman D.L., Tatsumi M., Gilson W.D., Ishimori T., Kedziorek D., Walczak P., Segars W.P., Chen H.H., Fritzges D., Izbudak I., et al., Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction, Circulation, 2000 112, 1451-1461.10.1161/CIRCULATIONAHA.105.537480Suche in Google Scholar PubMed PubMed Central

Received: 2016-6-3

Accepted: 2016-9-14

Published Online: 2016-12-26

Published in Print: 2016-1-1

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.