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Chromosomal variability of human mesenchymal stem cells cultured under hypoxic conditions.

Ueyama H, Horibe T, Hinotsu S, Tanaka T, Inoue T, Urushihara H, Kitagawa A, Kawakami K - J. Cell. Mol. Med. (2012)

Bottom Line: Bone marrow derived human mesenchymal stem cells (hMSCs) have attracted great interest from both bench and clinical researchers because of their pluripotency and ease of expansion ex vivo.However, these cells do finally reach a senescent stage and lose their multipotent potential.The safe, rapid expansion of hMSCs is critical for their clinical application.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Yoshidakonoecho, Sakyo-ku, Kyoto, Japan.

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Karyotyping analysis of hMSCs. (A) Morphology of cells in primary culture (left) and in late (13th) passage (right). The size of hMSCs in culture was enlarged with increasing passages. Scale bars, 200 μm. (B) G-banding karyotyping of cells with chromosomal anomalies (donor #100). Karyotyping analysis was performed as described in ‘Materials and methods’. The karyotype shows an addition on chromosome 12 [46,XY,add(12)(p13)]. The chro-mosomal anomaly is indicated by an arrow.
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fig01: Karyotyping analysis of hMSCs. (A) Morphology of cells in primary culture (left) and in late (13th) passage (right). The size of hMSCs in culture was enlarged with increasing passages. Scale bars, 200 μm. (B) G-banding karyotyping of cells with chromosomal anomalies (donor #100). Karyotyping analysis was performed as described in ‘Materials and methods’. The karyotype shows an addition on chromosome 12 [46,XY,add(12)(p13)]. The chro-mosomal anomaly is indicated by an arrow.

Mentions: Forty hMSC samples isolated from bone marrow of donors (16 men and 24 women) with a median age of 61 years (range 26–77 years) were analysed (Table S1). Bone marrow derived hMSCs attached to the culture surface and showed a fibroblast-like appearance in both normoxic and hypoxic cultures, and radial axons were enlarged with apparent directional arrangement with increasing passages (Fig. 1A). The size of hMSCs also increased in later passages. With a single counting of chromosome numbers following Giemsa staining, the normal human karyotype was observed, written as 46,XY or 46,XX. We next examined karyotypes to confirm that these cells maintained their chromosomal stability after in vitro expansion. Abnormal karyotypes from detailed karyotypic analysis are summarized in Table 1. Anomaly karyotypes were not detected on the other samples. For example, karyotypic analysis of hMSCs from donor #100 cultured under normoxic conditions was done at passages 1 (P1), 3 (P3) and 6 (P6), and abnormal karyotypes were detected at each passage. Some donors including #118 and #127, had chromosomal aberrations at early passage but not later passage. It is possible that abnormal karyotypes were detected at early stage and not at next stage, because 50 cells were sampled at random from each sample and karyotypic analysis was conducted on these cells in this study. When hMSCs with anomaly karyotypes are detected from early to later stage, it is more likely that the hMSCs have abnormal karyotypes. Numerical aberrations (e.g. aneuploidy) and structural aberrations (e.g. additions, deletions, inversions and translocations) were detected under both culture conditions (Table 1). Whereas insertions and translocations are detected frequently, relatively fewer deletions, resulting in simple inactivation of the associated genes, were observed (Table 1). For instance, one of the observed anomalous karyotypes was an addition on chromosome 12 [46,XY, add(12)(p13)], indicating that a portion of another chromosome was added to the short-arm p13 site of the right-hand chromosome 12 shown in Figure 1B. In terms of aneuploidy, gain and loss of sex chromosomes was detected frequently. Donor #104 had the karyotype inv(9)(p12q13), which is the most common pericentric inversion in human beings, with a frequency of 1–2.5% in the general population [23], and is considered a normal polymorphism.


Chromosomal variability of human mesenchymal stem cells cultured under hypoxic conditions.

Ueyama H, Horibe T, Hinotsu S, Tanaka T, Inoue T, Urushihara H, Kitagawa A, Kawakami K - J. Cell. Mol. Med. (2012)

Karyotyping analysis of hMSCs. (A) Morphology of cells in primary culture (left) and in late (13th) passage (right). The size of hMSCs in culture was enlarged with increasing passages. Scale bars, 200 μm. (B) G-banding karyotyping of cells with chromosomal anomalies (donor #100). Karyotyping analysis was performed as described in ‘Materials and methods’. The karyotype shows an addition on chromosome 12 [46,XY,add(12)(p13)]. The chro-mosomal anomaly is indicated by an arrow.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823094&req=5

fig01: Karyotyping analysis of hMSCs. (A) Morphology of cells in primary culture (left) and in late (13th) passage (right). The size of hMSCs in culture was enlarged with increasing passages. Scale bars, 200 μm. (B) G-banding karyotyping of cells with chromosomal anomalies (donor #100). Karyotyping analysis was performed as described in ‘Materials and methods’. The karyotype shows an addition on chromosome 12 [46,XY,add(12)(p13)]. The chro-mosomal anomaly is indicated by an arrow.
Mentions: Forty hMSC samples isolated from bone marrow of donors (16 men and 24 women) with a median age of 61 years (range 26–77 years) were analysed (Table S1). Bone marrow derived hMSCs attached to the culture surface and showed a fibroblast-like appearance in both normoxic and hypoxic cultures, and radial axons were enlarged with apparent directional arrangement with increasing passages (Fig. 1A). The size of hMSCs also increased in later passages. With a single counting of chromosome numbers following Giemsa staining, the normal human karyotype was observed, written as 46,XY or 46,XX. We next examined karyotypes to confirm that these cells maintained their chromosomal stability after in vitro expansion. Abnormal karyotypes from detailed karyotypic analysis are summarized in Table 1. Anomaly karyotypes were not detected on the other samples. For example, karyotypic analysis of hMSCs from donor #100 cultured under normoxic conditions was done at passages 1 (P1), 3 (P3) and 6 (P6), and abnormal karyotypes were detected at each passage. Some donors including #118 and #127, had chromosomal aberrations at early passage but not later passage. It is possible that abnormal karyotypes were detected at early stage and not at next stage, because 50 cells were sampled at random from each sample and karyotypic analysis was conducted on these cells in this study. When hMSCs with anomaly karyotypes are detected from early to later stage, it is more likely that the hMSCs have abnormal karyotypes. Numerical aberrations (e.g. aneuploidy) and structural aberrations (e.g. additions, deletions, inversions and translocations) were detected under both culture conditions (Table 1). Whereas insertions and translocations are detected frequently, relatively fewer deletions, resulting in simple inactivation of the associated genes, were observed (Table 1). For instance, one of the observed anomalous karyotypes was an addition on chromosome 12 [46,XY, add(12)(p13)], indicating that a portion of another chromosome was added to the short-arm p13 site of the right-hand chromosome 12 shown in Figure 1B. In terms of aneuploidy, gain and loss of sex chromosomes was detected frequently. Donor #104 had the karyotype inv(9)(p12q13), which is the most common pericentric inversion in human beings, with a frequency of 1–2.5% in the general population [23], and is considered a normal polymorphism.

Bottom Line: Bone marrow derived human mesenchymal stem cells (hMSCs) have attracted great interest from both bench and clinical researchers because of their pluripotency and ease of expansion ex vivo.However, these cells do finally reach a senescent stage and lose their multipotent potential.The safe, rapid expansion of hMSCs is critical for their clinical application.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Yoshidakonoecho, Sakyo-ku, Kyoto, Japan.

Show MeSH
Related in: MedlinePlus