|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2008) 32, 950958 (Printed in Great Britain)
Proliferation, multipotency and neuronal differentiation of cryopreserved neural progenitor cells derived from the olfactory neuroepithelium of the adult rat
Tao Xuea, Li Qiaoa, Jianhua Qiua*, Lianjun Lua, Dingjun Zhaa and Li Weib
aDepartment of Otolaryngology, Xijing Hospital, the Fourth Military Medical University, Xi‘an 710032, PR China
bDepartment of Obstetrics and Gynecology, Xijing Hospital, the Fourth Military Medical University, Xi‘an 710032, PR China
The use of olfactory neuroepithelium neural progenitor cells for transplantation has attracted attention in the treatment of many neurological disorders, which require efficient recovery methods and cryopreservation procedures. The purpose of this study was to evaluate different cryopreservation techniques for neural progenitor cells derived from the olfactory neuroepithelium (ONe NPCs) in adult rats. Initially, we compared the survival rates of cryopreserved ONe NPCs treated with six different cryoprotectants: dimethylsulfoxide (DMSO), ethylene glycol (EG) and glycerol, each with or without 10% FBS and with two different storage periods in liquid nitrogen (−196
Keywords: Neural precursor cell, Cryopreservation, Olfactory neuroepithelium.
*Corresponding author. Tel.: +86 29 84775381; fax: +86 29 83224744.
Stem cells are undifferentiated cells that have the ability to undergo numerous divisions and self-renewal in culture; in addition, they are able to differentiate into multilineage, functionally specialized cells. The replacement of lost or damaged neurons by neural stem cells (NSCs) or neural progenitor cells(NPCs) represents great promise for clinical treatment of many neurological disorders (Chiasson et al., 1999; Loeffler and Roeder, 2002; Weiss et al., 1996). NSCs, with their capacity for unlimited self renewal and production of non-restricted lineage- committed progenitors in contrast to neural ‘progenitor’ or ‘precursor’ cells, have long been thought of as central to the repair and regeneration processes of replacing cells lost in many devastating diseases. Multipotent NSCs can be found in the embryonic, neonatal and adult mammalian CNS (Reynolds and Weiss, 1992). The forebrain subventricular zone (SVZ) and dentate gyrus are considered to be the major sources of self-renewing, multipotent NSCs. Furthermore, multipotential precursors with stem cell features can be isolated not only from the SVZ, but also from the entire rostral extension, including the distal portion within the olfactory bulb (OB) (Gritti et al., 2002; Otaegi et al., 2007).
Recently, the regenerative capacity of the olfactory system has attracted attention. Olfactory ensheathing cells (OECs) from adult OBs have been used to provide limited axonal regeneration (Dombrowski et al., 2006; Lu et al., 2006; Ramer et al., 2004) and to repair demyelinated regions of the CNS (Lakatos et al., 2000; Nieto-Sampedro, 2003; Santos-Benito and Ramon-Cueto, 2003). However, the harvest of OECs from the olfactory bulb involves highly invasive surgery, making this a problematic source for clinical therapy (Barnett et al., 2000; Deng et al., 2006; Lopez-Vales et al., 2007; Lu et al., 2002). The olfactory neuroepithelium (ONe) undergoes lifelong repair by progenitors, which are capable of replacing both neuronal and supporting cells (Beites et al., 2005; Calof and Chikaraishi, 1989; Hahn et al., 2005). Previously, some authors (Beites et al., 2005; Chuah et al., 1991) developed procedures for isolation and culture of ONe-derived neurosphere-forming cells from adult rats that remain mitotically active and have the characteristics of neural progenitor cells (NPCs). From a developmental standpoint, murine ONe NPCs represent an accessible and important system for the study of basic ONe NPC properties such as self-renewal and multipotency. The clinical implications of ONe NPCs are potentially profound. Cryopreservation is a prerequisite for quality assurance, storage and distribution required for tissue that shall be used clinically. Therefore, development of appropriate cryopreservation techniques is required. Cryopreservation of NSCs has been done using dimethyl sulfoxide (DMSO). However, the role of different cryoprotectants in the preservation of ONe NPCs has not been studied in detail. Successful long-term storage and preservation of ONe NPCs is an important prerequisite for their potential therapeutic application in regenerative approaches such as transplantation.
We believe that ONe NPCs may be an ideal source for autologous NSC transplantation and will eventually be used clinically. The widespread use of various treatment protocols using ONe NPCs have resulted in an increased demand for cryobiological techniques for storing these cells. The need has led to a re-examination of many cryobiological practices. However, DMSO is known to be toxic, with side-effects after prolonged exposure (Kim et al., 2007; Syme et al., 2004). Therefore, developing a cryopreservation medium with little toxicity is mandatory. In this paper we show that ONe NPC cultures derived from the adult rat can be cryopreserved. This enables the investigator to produce large numbers of NPCs from a small starting population of ONe NPCs and freeze these NPCs for later use. The ability to cryopreserve ONe NPCs greatly simplifies the use of this cell culture system.
2 Materials and methods
2.1 Animals and reagents
Male and female Sprague–Dawley rats, 200–250
Fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) were purchased from PeproTech (USA) and B27 was purchased from Invitrogen (CA, USA). The CCK-8 kit was purchased from Dojindo Laboratories (Japan). Others reagents for tissue culture were purchased from HyClone (USA) and Sigma (USA).
2.2 Removal of the olfactory neuroepithelium tissue
On the first day of the experiment, rats were anesthetized by an i.p. injection of chloral hydrate 0.3
A midline skin incision was made in the nasal bone and a burr hole was drilled. A piece of tissue including the olfactory neuroepithelium (white arrowheads) was transferred into phosphate-buffered saline (PBS) and cut into small pieces under a surgical microscope (A). Three days after plating, some rounded cells started to divide. Scale bar corresponds to 100
2.3 Olfactory neuroepithelium NPC culture medium
ONe NPCs were seeded in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) with heat-inactivated 10% fetal bovine serum (FBS) (Invitrogen Cat No. 10099-141) containing 0.6% glucose, 1:50 B27, 2
To initiate differentiation, after FGF-2 and EGF were removed, colonies of ONe NPCs were plated for 10
2.4 Cryopreservation techniques and post-thawed
For the cryopreservation of ONe NPCs populations, cells were prepared as described above. After at least two passages of neurospheres, the samples were resuspended in special cryoprotectants, stored in a freezer at −70
2.5 Measurement of cell viability and cell proliferation assay
To measure trypan blue exclusion as described by Milosevic et al. (2005), fresh or frozen-thawed ONe NPCs were incubated in triplicates in a 0.25% dye solution (Gibco). Total cell numbers and the number of trypan blue positive cells was then determined by counting at least 200 cells per sample in a hemocytometer. Cell viability (percentage) is the ratio of the number of trypan blue-impermeable cells to the total cell count (trypan blue-impermeable cell number/total cell number). Cells were observed under a microscope and counted as stained and nonstained cells on hemocytometer separately, then the viable cell ratios were calculated according to the following formula: viable cell ratio (%)
To measure CCK-8, fresh or frozen-thawed ONe NPCs in the above, different cryoprotective additives were monitored via an inverted microscope and their viability was detected every 48
2.6 Clonogenic survival assay
ONe NPC survival after exposure to the freezing process with different cryoprotectants was measured via the colony-forming assay as described by Davis and Temple (1994). Clonogenicity was determined by measuring colony formation as described by Lu and Wong (2005) and Zappone et al. (2000). In brief, after the cells were thawed and washed, ONe NPCs were adjusted to a final concentration of 5
2.7 BrdU labeling and neurosphere differentiation
To label the cells as described by Namba et al. (2007) and Lei et al. (2007), 6
2.8 Immunocytochemistry and immunofluorescence
The expression of each antigen was examined in separate experiments at least three times. After each treatment, cells were fixed with 4% paraformaldehyde for 15
For the BrdU/NeuN or BrdU/GFAP immunofluorescent double-labeling, the fixed cells were incubated in 2N HCl at 37
All statistical analyses were performed using the SPSS 11.0 (Jandel Corp, San Rafael, CA, USA). Results are expressed as mean
3.1 Isolation and characterization of ONe NPCs
To isolate ONe NPCs, a cell suspension was prepared from adult rat olfactory neuroepithelium tissue and plated under tissue culture conditions in the presence of FGF-2 and EGF. Three days after plating, some rounded cells started to divide (Fig. 1B, black arrowheads). A smaller proportion of the cells differentiated into neuron- like cells (Fig. 1C, black arrowheads). Daily inspection of the cultures showed that the rounded cells divided, forming cell aggregates or spheres (Moses et al., 2006; Reynolds and Weiss, 1996), reaching confluence in the presence of the mitogens by the 7th day (Fig. 1D).
Under these standard growth conditions, cells could be passaged every 7–9
Immunocytochemistry analyses revealed that ONe NPCs formed neurospheres. ONe NPCs formed neurospheres expressed nestin. Scale bar corresponds to 50
3.2 Viability of cryopreserved ONe NPCs
Necrotic cell death caused by the cryopreservation process was measured by the uptake of trypan blue immediately after thawing and washing out cryoprotective agents. Cells with compromised plasma membranes permitted entry of the dye, whereas viable cells excluded the dye. After counting, we observed necrotic cell death was below 30% in most cases (Fig. 3A). Samples frozen for 3
Before freezing, ONe NPCs were expanded as a monolayer (see Section
Cryopreserved and thawed ONe NPC recovery was assessed by traditional assay of colony formation. Viable single cells were grown, generating colonies. For each freeze–thaw sample, a value for total CFUs generated within 2
3.3 Differentiation of cryopreserved treated ONe NPCs
Within the first week of the post-thawing period, ONe NPCs formed neurospheres identical to those seen in the fresh tissue (Fig. 4G). After removal of growth factors, cryopreserved and thawed neurospheres readily differentiated into neurons (NeuN-positive), astrocytes (GFAP-positive), as detected by Immunofluorescent double labeling (Fig. 4A–F). The relative number of neurons was estimated by calculating the percentage of NeuN-positive vs. BrdU-labeled cells. On the one hand, the number of viable cells was markedly reduced after the freeze–thaw process. However, on the other hand, the relative number of neurons in the cryopreserved and thawed tissue showed no statistical difference from the fresh untreated one: 8.26
Neural differentiation of ONe NPCs after thawing. Frozen-thawed ONe NPCs were labeled by BrdU for 3
Successful long-term storage and preservation of stem cells is an important precondition for their potential therapeutic application in regenerative approaches such as transplantation. The use of cryopreserved NSCs/NPCs in gene and cell therapy also requires preservation of their differentiation and proliferative capacities. In this light, it is necessary to assess whether the process of cryopreservation affects either the proliferative capacity of NSCs/NPCs or their developmental potential. The aim of various cryopreservation procedures is to minimize cell injury during the freeze–thaw process. Cell injury may result from extensive cellular dehydration (‘solution effect’) and/or intracellular ice crystallization (‘mechanical cell damage’) (Mazur, 1966; Meryman, 1956; Zhao et al., 2001). Today, the cryopreservation of adult human bone marrow mesenchymal stem cells (hMSC) is considered to offer not only a population of pluripotential cells but also an accessible means for establishing an abundant hMSC reservoir for further experimentation (Kotobuki et al., 2005; Xiang et al., 2007). Indeed, the different storage conditions used for NSCs/NPCs show a difference in cost and availability. Thus, the search for optimal freezing and storing procedures is a pivotal prerequisite for the general and systematic application of these cells in both preclinical and clinical settings. Similarly, with cryopreservation yields of 50–60% for HSCs, which are widely used clinically (Querol et al., 2000; Yang et al., 2003), our study demonstrates that deep-freezing at −196
Frozen cells are injured by the direct effect of low temperature and the formation of ice crystals. On the one hand, intracellular ice formation coupled with a high rate of cooling may rupture the cell; on the other hand, extracellular ice formation results in increased extracellular medium osmolality as water is taken up, causing severe dehydration (Balint et al., 1999; Broxmeyer et al., 2003; Buchanan et al., 2004; Meyer et al., 2006). On the other hand, rate-controlled cryopreservation reduces this potential damage (Zhao et al., 2001). Although our results indicated that both techniques performed equally well, rate-controlled freezing should be explored in further studies.
If the freezing procedure is optimal and no cell damage has occurred, no further damage would be expected during the storage period (Katayama et al., 1997). For long-term cryopreservation, however, it is necessary for the storage temperature to be low enough to block all enzymatic pathways and metabolism of the cells. Our trypan-blue exclusion and colony-forming assay indicated that there was no statistical difference between the mean cell recovery rate after cryopreservation in liquid nitrogen for 3
The critical issue of cryopreservation is whether frozen-thawed ONe NPCs are able to proliferate and to differentiate into neurons, astrocytes and oligodendrocytes (Hancock et al., 2000; Milosevic et al., 2005). Our objectives were to analyze the effect of cryopreservation on the survival of ONe NPCs and to establish whether cryopreserved ONe NPCs are able to proliferate in tissue culture while maintaining their proliferation and multipotency activity. The results obtained in this study indicate no statistically significant difference in ONe NPC multipotency activity between fresh cells and frozen-thawed cells. Furthermore, we obtained the same survival rate in the six methods studied. The neural phenotype of the cells which differentiated from cryopreserved ONe NPCs was estimated by immunofluorescence assay of neuron markers (NeuN) and astrocytes markers (GFAP). A previous study by See et al. (2004) showed that GalC was reported to rapidly disappear from the surface of cultured oligodendrocytes; our immunocytochemistry assay and other studies (Othman et al., 2005) have all proved that the ability of the cells to differentiate into oligodendrocytes was lower than their ability to differentiate into neurons or astrocytes, and only a minority of them were GalC positive. Thus, it was very difficult to get a positive result in the immunofluorescence assay and the cells were not estimated by the immunofluorescence assay of GlaC. Upon withdrawal of EGF and FGF-2, the thawed ONe NPCs cultures differentiated into neuronal cultures which showed the same overall appearance as the ones untreated by cryopreservation.
We have shown that ONe NPCs can be cryopreserved for long periods. Moreover, we have demonstrated that cryopreserved ONe NPCs can differentiate into neural cells which expressed neuron-specific gene products. In conclusion, our study describes excellent cryopreservation techniques for ONe NPCs which may not only preserve cell proliferation properties, but also maintain the multi-differentiative potential. It is tempting to speculate that we have found an excellent way for preparing tissue used for stem cell restorative therapy which meets the necessary safety and quality control standards.
This work was supported by grants to Prof. Jianhua Qiu from the National Natural Science Foundation of China (No. 30572023).
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Received 14 December 2007/19 February 2008; accepted 2 April 2008doi:10.1016/j.cellbi.2008.04.012