Side population is not necessary or sufficient for a cancer stem cell phenotype in glioblastoma multiforme

There is strong evidence for the existence of cancer stem cells (CSCs) in the aggressive brain tumor glioblastoma multiforme (GBM). These cells have stem-like self-renewal activity and increased tumor initiation capacity and are believed to be responsible for recurrence due to their resistance to therapy. Several techniques have been used to enrich for CSC, including growth in serum-free defined media to induce sphere formation, and isolation of a stem-like cell using exclusion of the fluorescent dye Hoechst 33342, the side population (SP). We show that sphere formation in GBM cell lines and primary GBM cells enriches for a CSC-like phenotype of increased self-renewal gene expression in vitro and increased tumor initiation in vivo. However, the SP was absent from all sphere cultures. Direct isolation of the SP from the GBM lines did not enrich for stem-like activity in vitro, and tumor-initiating activity was lower in sorted SP compared with non-SP and parental cells. Transient exposure to doxorubicin enhanced both CSC and SP frequency. However, doxorubicin treatment altered the cytometric profile and obscured the SP demonstrating the difficulty of identifying SP in cells under stress. Doxorubicin-exposed cells showed a transient increase in SP, but the doxorubicin-SP cells were still not enriched for a stem-like self-renewal phenotype. These data demonstrate that the GBM SP does not necessarily contribute to self-renewal or tumor initiation, key properties of a CSC, and we advise against using SP to enumerate or isolate CSC.     

Tumorigenesis of chemotherapeutic drug-resistant cancer stem-like cells in brain glioma

Glioblastoma multiforme (GBM) is the most frequently occurring brain cancer. Although the existence of cancer stem cells (CSCs) in GBM has been established, there is little evidence to explain the link between CSCs and chemoresistance. In this study, we developed a dissociated cell system of human GBM cells, A172 and established GBM2 cells, that have shown resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). After exposure to a lethal dose of BCNU, the small population of GBM cancer cells survived and proliferated, as opposed to direct inhibition of the apoptosis and activation of the proliferation signal. Also, these cells contained subpopulations of stem-like cells, expressing CD133, CD117, CD90, CD71, and CD45 cell-surface markers, and had the capacity for multipotency. Moreover, we observed that BCNU-resistant subpopulations derived from GBM cancer cells can be grown to tumors when transplanted into severe combined immunodeficient (SCID) mouse brain. These results demonstrated that BCNU-resistant subpopulations derived from GBM have cancer stem-like cell properties. These findings provide further evidence that CSCs in GBM display chemotherapeutic drug resistance. Hopefully, it will be possible to improve the therapeutic outcome of GBM, leading to better anticancer strategies.     

Persistence of CD133+ cells in human and mouse glioma cell lines: detailed characterization of GL261 glioma cells with cancer stem cell-like properties

The concept of cancer stem cells suggests that there are malignant stem-like cells within a tumor that are responsible for tumor renewal and resistance to cytotoxic therapies. Studies have identified glioma stem-like cells that extrude Hoechst 33342 dye, representing a double-negative "side population" (SP) thought to be selectively resistant to drug therapy. A CD133+ stem cell-like subpopulation has been isolated from a human glioma that was enriched for tumor-initiating cells. It is unknown whether CD133+ cells with similar phenotype persist in established glioma cell lines, or if CD133 is a marker of glioma stem-like cells in rodents. We investigated whether CD133+ and SP cells existed in the GL261 cell line, a syngeneic mouse glioma model that is widely used for preclinical and translational research. Intracerebral injection of less than 100 CD133+ GL261 cells formed tumors, whereas it required 10,000 CD133(-) cells to initiate a tumor. CD133+ GL261 cells expressed nestin, formed tumor spheres with high frequency, and differentiated into glial and neuronal-like cells. Similar to GL261, seven human glioma cell lines analyzed also contained a rare CD133+ population. Surprisingly, we found that CD133+ GL261 cells did not reside in the SP, nor did the majority ( approximately 94%) of CD133+ human glioma cells. These results demonstrate that the expression of CD133 in murine glioma cells is associated with enhanced tumorigenicity and a stem-like phenotype. This study also reveals a previously unrecognized level of heterogeneity in glioma cell lines, exposing several populations of cells that have characteristics of cancer stem cells.     

Clinical and biological implications of CD133-positive and CD133-negative cells in glioblastomas

A number of recent reports have demonstrated that only CD133-positive cancer cells of glioblastoma multiforme (GBM) have tumor-initiating potential. These findings raise an attractive hypothesis that GBMs can be cured by eradicating CD133-positive cancer stem cells (CSCs), which are a small portion of GBM cells. However, as GBMs are known to possess various genetic alterations, GBMs might harbor heterogeneous CSCs with different genetic alterations. Here, we compared the clinical characteristics of two GBM patient groups divided according to CD133-positive cell ratios. The CD133-low GBMs showed more invasive growth and gene expression profiles characteristic of mesenchymal or proliferative subtypes, whereas the CD133-high GBMs showed features of cortical and well-demarcated tumors and gene expressions typical of proneuronal subtype. Both CD133-positive and CD133-negative cells purified from four out of six GBM patients produced typical GBM tumor masses in NOD-SCID brains, whereas brain mass from CD133-negative cells showed more proliferative and angiogenic features compared to that from CD133-positive cells. Our results suggest, in contrast to previous reports that only CD133-positive cells of GBMs can initiate tumor formation in vivo CD133-negative cells also possess tumor-initiating potential, which is indicative of complexity in the identification of cancer cells for therapeutic targeting.     

Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma

Brain tumors can arise following deregulation of signaling pathways normally activated during brain development and may derive from neural stem cells. Given the requirement for Hedgehog in non-neoplastic stem cells, we investigated whether Hedgehog blockade could target the stem-like population in glioblastoma multiforme (GBM). We found that Gli1, a key Hedgehog pathway target, was highly expressed in 5 of 19 primary GBM and in 4 of 7 GBM cell lines. Shh ligand was expressed in some primary tumors, and in GBM-derived neurospheres, suggesting a potential mechanism for pathway activation. Hedgehog pathway blockade by cyclopamine caused a 40%-60% reduction in growth of adherent glioma lines highly expressing Gli1 but not in those lacking evidence of pathway activity. When GBM-derived neurospheres were treated with cyclopamine and then dissociated and seeded in media lacking the inhibitor, no new neurospheres formed, suggesting that the clonogenic cancer stem cells had been depleted. Consistent with this hypothesis, the stem-like fraction in gliomas marked by both aldehyde dehydrogenase activity and Hoechst dye excretion (side population) was significantly reduced or eliminated by cyclopamine. In contrast, we found that radiation treatment of our GBM neurospheres increased the percentage of these stem-like cells, suggesting that this standard therapy preferentially targets better-differentiated neoplastic cells. Most importantly, viable GBM cells injected intracranially following Hedgehog blockade were no longer able to form tumors in athymic mice, indicating that a cancer stem cell population critical for ongoing growth had been removed. Disclosure of potential conflicts of interest is found at the end of this article.     

Lectins identify glycan biomarkers on glioblastoma-derived cancer stem cells

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. Despite aggressive therapy with surgery, radiotherapy, and chemotherapy, nearly all patients succumb to disease within 2 years. Several studies have supported the presence of stem-like cells in brain tumor cultures that are CD133-positive, are capable of self-renewal, and give rise to all cell types found within the tumor, potentially perpetuating growth. CD133 is a widely accepted marker for glioma-derived cancer stem cells; however, its reliability has been questioned, creating a need for other identifiers of this biologically important subpopulation. We used a panel of 20 lectins to identify differences in glycan expression found in the glycocalyx of undifferentiated glioma-derived stem cells and differentiated cells that arise from them. Fluorescently labeled lectins that specifically recognize α-N-acetylgalactosamine (GalNAc) and α-N-acetylglucosamine (GlcNAc) differentially bound to the cell surface based on the state of cellular differentiation. GalNAc and GlcNAc were highly expressed on the surface of undifferentiated cells and showed markedly reduced expression over a 12-day duration of differentiation. Additionally, the GalNAc-recognizing lectin Dolichos biflorus agglutinin was capable of specifically selecting and sorting glioma-derived stem cell populations from an unsorted tumor stock and this subpopulation had proliferative properties similar to CD133(+) cells in vitro and also had tumor-forming capability in vivo. Our preliminary results on a single cerebellar GBM suggest that GalNAc and GlcNAc are novel biomarkers for identifying glioma-derived stem cells and can be used to isolate cancer stem cells from unsorted cell populations, thereby creating new cell lines for research or clinical testing.     

Hedgehog signaling regulates brain tumor-initiating cell proliferation and portends shorter survival for patients with PTEN-coexpressing glioblastomas

The identification of brain tumor stem-like cells (BTSCs) has implicated a role of biological self-renewal mechanisms in clinical brain tumor initiation and propagation. The molecular mechanisms underlying the tumor-forming capacity of BTSCs, however, remain unknown. Here, we have generated molecular signatures of glioblastoma multiforme (GBM) using gene expression profiles of BTSCs and have identified both Sonic Hedgehog (SHH) signaling-dependent and -independent BTSCs and their respective glioblastoma surgical specimens. BTSC proliferation could be abrogated in a pathway-dependent fashion in vitro and in an intracranial tumor model in athymic mice. Both SHH-dependent and -independent brain tumor growth required phosphoinositide 3-kinase-mammalian target of rapamycin signaling. In human GBMs, the levels of SHH and PTCH1 expression were significantly higher in PTEN-expressing tumors than in PTEN-deficient tumors. In addition, we show that hyperactive SHH-GLI signaling in PTEN-coexpressing human GBM is associated with reduced survival time. Thus, distinct proliferation signaling dependence may underpin glioblastoma propagation by BTSCs. Modeling these BTSC proliferation mechanisms may provide a rationale for individualized glioblastoma treatment.     

Isolation and characterization of a population of stem-like progenitor cells from an atypical meningioma

The majority of meningiomas are benign tumors associated with favorable outcomes; however, the less common aggressive variants with unfavorable outcomes often recur and may be due to subpopulations of less-differentiated cells residing within the tumor. These subpopulations of tumor cells have tumor-initiating properties and may be isolated from heterogeneous tumors when sorted or cultured in defined medium. We report the isolation and characterization of a population of tumor-initiating cells derived from an atypical meningioma. We identify a tumor-initiating population from an atypical meningioma, termed meningioma-initiating cells (MICs). These MICs self-renew, differentiate, and can recapitulate the histological characteristics of the parental tumor when transplanted at 1000 cells into the flank regions of athymic nude mice. Immunohistochemistry reveals stem-like protein expression patterns similar to neural stem and progenitor cells (NSPCs) while genomic profiling verified the isolation of cancer cells (with defined meningioma chromosomal aberrations) from the bulk tumor. Microarray and pathway analysis identifies biochemical processes and gene networks related to aberrant cell cycle progression, particularly the loss of heterozygosity of tumor suppressor genes CDKN2A (p16^INK4A), p14^ARF, and CDKN2B (p15^INK4B). Flow cytometric analysis revealed the expression of CD44 and activated leukocyte adhesion molecule (ALCAM/CD166); these may prove to be markers able to identify this cell type. The isolation and identification of a tumor-initiating cell population capable of forming meningiomas demonstrates a useful model for understanding meningioma development. This meningioma model may be used to study the cell hierarchy of meningioma tumorogenesis and provide increased understanding of malignant progression.     

EGCG inhibits the growth and tumorigenicity of nasopharyngeal tumor-initiating cells through attenuation of STAT3 activation

A subset of cancer cells, termed cancer stem cells (CSCs) or tumor-initiating cells (TICs) could initiate tumors and are responsible for tumor recurrence and chemotherapeutic resistance. In this study, we enriched TICs in nasopharyngeal carcinoma (NPC) by the spheres formation and characterized the stem-like signatures such as self-renewal, proliferation, chemoresistance and tumorigenicity. By this method, we investigated that epigallocathechin gallate (EGCG), the major polyphenol in green tea could target TICs and potently inhibit sphere formation, eliminate the stem-like properties and enhance chemosensitivity in NPC through attenuation of STAT3 activation, which could be important in regulating the stemness expression in NPC. Our results demonstrated that STAT3 pathway plays an important role in mediating tumor-initiating capacities in NPC and suggest that inactivation of STAT3 with EGCG may represent a potential preventive and therapeutic approach for NPC.     

Emerging targets for glioblastoma stem cell therapy

Glioblastoma multiforme (GBM), designated as World Health Organization (WHO) grade IV astrocytoma, is a lethal and therapy-resistant brain cancer comprised of several tumor cell subpopulations, including GBM stem cells (GSCs) which are believed to contribute to tumor recurrence following initial response to therapies. Emerging evidence demonstrates that GBM tumors are initiated from GSCs. The development and use of novel therapies including small molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of GSCs, immunotherapy, and non-coding microRNAs may provide better means of treating GBM. Identification and characterization of GSC-specific signaling pathways would be necessary to identify specific therapeutic targets which may lead to the development of more efficient therapies selectively targeting GSCs. Several signaling pathways including mTOR, AKT, maternal embryonic leucine zipper kinase (MELK), NOTCH1 and Wnt/b-catenin as well as expression of cancer stem cell markers CD133, CD44, Oct4, Sox2, Nanog, and ALDH1A1 maintain GSC properties. Moreover, the data published in the Cancer Genome Atlas (TCGA) specifically demonstrated the activated PI3K/AKT/mTOR pathway in GBM tumorigenesis. Studying such pathways may help to understand GSC biology and lead to the development of potential therapeutic interventions to render them more sensitive to chemotherapy and radiation therapy. Furthemore, recent demonstration of dedifferentiation of GBM cell lines into CSC-like cells prove that any successful therapeutic agent or combination of drugs for GBM therapy must eliminate not only GSCs, but the differentiated GBM cells and the entire bulk of tumor cells.     

miR-182 integrates apoptosis,growth, and differentiation programs in glioblastoma

Glioblastoma multiforme (GBM) is a lethal, therapy-resistant brain cancer consisting of numerous tumor cell subpopulations, including stem-like glioma-initiating cells (GICs), which contribute to tumor recurrence following initial response to therapy. Here, we identified miR-182 as a regulator of apoptosis, growth, and differentiation programs whose expression level is correlated with GBM patient survival. Repression of Bcl2-like12 (Bcl2L12), c-Met, and hypoxia-inducible factor 2α (HIF2A) is of central importance to miR-182 anti-tumor activity, as it results in enhanced therapy susceptibility, decreased GIC sphere size, expansion, and stemness in vitro. To evaluate the tumor-suppressive function of miR-182 in vivo, we synthesized miR-182-based spherical nucleic acids (182-SNAs); i.e., gold nanoparticles covalently functionalized with mature miR-182 duplexes. Intravenously administered 182-SNAs penetrated the blood–brain/blood–tumor barriers (BBB/BTB) in orthotopic GBM xenografts and selectively disseminated throughout extravascular glioma parenchyma, causing reduced tumor burden and increased animal survival. Our results indicate that harnessing the anti-tumor activities of miR-182 via safe and robust delivery of 182-SNAs represents a novel strategy for therapeutic intervention in GBM.     

Spontaneous in vitro transformation of adult neural precursors into stem-like cancer cells

Recent studies have found that cellular self-renewal capacity in brain cancer is heterogeneous, with only stem-like cells having this property. A link between adult stem cells and cancer stem cells remains, however, to be shown. Here, we describe the emergence of cancer stem-like cells from in vitro cultured brain stem cells. Adult rat subventricular zone (SVZ) stem cells transformed into tumorigenic cell lines after expansion in vitro . These cell lines maintained characteristic features of stem-like cells expressing Nestin, Musashi-1 and CD133, but continued to proliferate upon differentiation induction. Karyotyping detected multiple acquired chromosomal aberrations, and syngeneic transplantation into the brain of adult rats resulted in malignant tumor formation. Tumors revealed streak necrosis and displayed a neural as well as an undifferentiated phenotype. Deficient downregulation of platelet-derived growth factor (PDGF) receptor alpha was identified as candidate mechanism for tumor cell proliferation, and its knockdown by siRNA resulted in a reduction of cell growth. Our data point to adult brain precursor cells to be transformed in malignancies. Furthermore, in vitro expansion of adult neural stem cells, which will be mandatory for therapeutic strategies in neurological disorders, also harbors the risk for amplifying precursor cells with acquired genetic abnormalities and induction of malignant tumors after transplantation.     

Cryopreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell--biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate-fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell-biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreservation of "ready-to-use" cell-biomaterial constructs.     

Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells

The rat 9L gliosarcoma is a widely used syngeneic rat brain tumor model that closely simulates glioblastoma multiforme when implanted in vivo. In this study, we sought to isolate and characterize a subgroup of cancer stem-like cells (CSLCs) from the 9L gliosarcoma cell line, which may represent the tumor-initiating subpopulation of cells. We demonstrate that these CSLCs form clonal-derived spheres in media devoid of serum supplemented with the mitogens epidermal growth factor and basic fibroblast growth factor, express the NSC markers Nestin and Sox2, self-renew, and differentiate into neuron-like and glial cells in vitro. More importantly, these cells can propagate and recapitulate tumors when implanted into the brain of syngeneic Fisher rats, and they display a more aggressive course compared with 9L gliosarcoma cells grown in monolayer cultures devoid of mitogens. Furthermore, we compare the chemosensitivity and proliferation rate of 9L gliosarcoma cells grown as a monolayer to those of cells grown as floating spheres and show that the sphere-generated cells have a lower proliferation rate, are more chemoresistant, and express several antiapoptosis and drug-related genes, which may prove to have important clinical implications. Disclosure of potential conflicts of interest is found at the end of this article.     

Spontaneous Neoplastic Transformation of WB-F344 Rat Liver Epithelial Cells

Several studies have shown that cultured rat liver epithelial cells transform spontaneously after chronic maintenance in a confluent state in vitro. In the present study, multiple independent lineages of low-passage WB-F344 rat liver epithelial stem-like cells were initiated and subjected in parallel to selection for spontaneous transformation to determine whether spontaneous acquisition of tumorigenicity was the result of events (genetic or epigenetic) that occurred independently and stochastically, or reflected the expression of a pre-existing alteration within the parental WB-F344 cell line. Temporal analysis of the spontaneous acquisition of tumorigenicity by WB-F344 cells demonstrated lineage-specific differences in the time of first expression of the tumorigenic phenotype, frequencies and latencies of tumor formation, and tumor differentiations. Although spontaneously transformed WB-F344 cells produced diverse tumor types (including hepatocellular carcinomas, cholangiocarcinomas, hepatoblastomas, and osteogenic sarcomas), individual lineages yielded tumors with consistent and specific patterns of differentiation. These results provide substantial evidence that the stochastic accumulation of independent transforming events during the selection regimen in vitro were responsible for spontaneous neoplastic transformation of WB-F344 cells. Furthermore, cell lineage commitment to a specific differentiation program was stable with time in culture and with site of transplantation. This is the first report of a cohort of related, but independent, rat liver epithelial cell lines that collectively produce a spectrum of tumor types but individually reproduce a specific tumor type. These cell lines will provide valuable reagents for investigation of the molecular mechanisms involved in the differentiation of hepatic stem-like cells and for examination of potential causal relationships in spontaneously transformed rat liver epithelial cell lines between molecular/cellular alterations and the ability to produce tumors in syngeneic animals.     

Virotherapy as an approach against cancer stem cells

It has been hypothesized that cancers originate from a small population of cells with stem cell-like characteristics, including self-renewal and pluripotency. Such tumor-initiating cells, also referred to as cancer stem cells, are thought to account for relapses following seemingly successful treatments, because their slow turnover and capacity for expelling anti-tumor drugs leaves them untouched by conventional treatment regimens. Targeting of cancer stem cells might be key for improving survival and producing cures in patients with metastatic tumors. Viruses enter cells though infection and might therefore not be sensitive to stem cell resistance mechanisms. During the last decades, oncolytic adenoviruses have been shown to effectively kill cancer cells, by seizing control of their DNA replication machinery and utilizing it for the production of new virions, ultimately resulting in the rupture of the cell. Human safety data in cancer trials has been excellent even when the dose of administered adenovirus has been high. Future approaches include additional modifications of the adenoviral genome that prime them to attack cancer stem cells specifically, utilizing linage-specific cell surface markers, dysfunctional stem cell signaling pathways or up-regulated oncogenic genes. However, already existing oncolytic adenoviruses have displayed potential to efficiently kill not only differentiated cancer cells, but also tumor-initiating stem cells. Here, we review the current literature that supports the existence of cancer stem cells and discuss the potential of virotherapy for killing tumor-initiating cells.     

Cytotoxic T lymphocytes efficiently recognize human colon cancer stem-like cells

Cancer stem-like cells (CSCs) and tumor-initiating cells (TICs) are a small population of cancer cells that share three properties: tumor initiating ability, self-renewal, and differentiation. These properties suggest that CSCs/TICs are essential for tumor maintenance, recurrence, and distant metastasis. Here, we show that cytotoxic T lymphocytes (CTLs) specific for the tumor-associated antigen CEP55 can efficiently recognize colon CSCs/TICs both in vitro and in vivo. Using Hoechst 33342 dye staining, we isolated CSCs/TICs as side population (SP) cells from colon cancer cell lines SW480, HT29, and HCT15. The SP cells expressed high levels of the stem cell markers SOX2, POU5F1, LGR5, and ALDH1A1 and showed resistance to chemotherapeutic agents such as irinotecan or etoposide.To evaluate the susceptibility of SP cells to CTLs, we used CTL clone 41, which is specific for the CEP55-derived antigenic peptide Cep55/c10orf3_193 (10) (VYVKGLLAKI). The SP cells expressed HLA class I and CEP55 at the same level as the main population cells. The SP cells were susceptible to CTL clone 41 at the same level as main population cells. Furthermore, adoptive transfer of CTL clone 41 inhibited tumor growth of SW480 SP cells in vivo. These observations suggest that Cep55/c10orf3_193(10) peptide-based cancer vaccine therapy or adoptive cell transfer of the CTL clone is a possible approach for targeting chemotherapy-resistant colon CSCs/TICs.     

Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification

This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48–72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.