Mesenchymal Stem Cells Isolated from the Human Bone Marrow: Cultivation, Phenotypic Analysis and Changes in Proliferation Kinetics

Summary: Human adult mesenchymal stem cells (MSCs) are rare elements living in various organs (e.g. bone marrow, skeletal muscle), with capability to differentiate in various cell types (e.g. chondrocytes, adipocytes and osteoblasts). In the year 2000, Gronthos and co-workers isolated stem cells from the human dental pulp (DPSCs). Later on, stem cells from exfoliated tooth were also obtained. The aims of our study were to establish protocol of DPSCs isolation and to cultivate DPSCs either from adult or exfoliated tooth, and to compare these cells with mesenchymal progenitor cell (MPCs) cultures. MPCs were isolated from the human bone marrow of proximal femur. DPSCs were isolated from deciduous and permanent teeth. Both cell types were cultivated under the same conditions in the media with 2 % of FCS supplemented with PDGF and EGF growth factors. We have cultivated undifferentiated DPSCs for long time, over 60 population doublings in cultivation media designed for bone marrow MPCs. After reaching Hayflick's limit, they still have normal karyotype. Initial doubling time of our cultures was from 12 to 50 hours for first 40 population doublings, after reaching 50 population doublings, doubling time had increased to 60-90 hours. Regression analysis of uncumulated population doublings proved tight dependence of population doublings on passage number and slow decrease of proliferation potential. In comparison with bone marrow MPCs, DPSCs share similar biological characteristics and stem cell properties. The results of our experiments proved that the DPSCs and MPCs are highly proliferative, clonogenic cells that can be expanded beyond Hayflick's limit and remain cytogenetically stable. Moreover we have probably isolated two different populations of DPSCs. These DPSCs lines differed one from another in morphology. Because of their high proliferative and differentiation potential, DPSCs can become more attractive, easily accessible source of adult stem cells for therapeutic purposes.

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“…Bone marrow mononuclear cells were obtained by optimized Ficoll-Paque density gradient centrifugation (11).…”

Section: Methodsmentioning

confidence: 99%

Human Dental Pulp Stem Cells – Isolation and Long Term Cultivation

Suchánek

Soukup

Ivančaková

et al. 2007

Acta Med. (Hradec Kralove, Czech Repub.)

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“…Independent laboratories reported contrasting results on the accumulation of chromosomal aberrations during in vitro culture, a debate that still remains open [Ben-David et al, 2011, 2012Sensebé et al, 2012]. Some authors showed that human bone marrow-derived MSCs remain chromosomally stable throughout long-term culture [Pittenger and Martin, 2004;Soukup et al, 2006;Bernardo et al, 2007;Zhang et al, 2007;Sensebé et al, 2012], whereas others claimed the occurrence of spontaneous numerical and structural chromosome aberrations within 10 passages of in vitro culture ( table 2 ). To this regard, monosomy of chromosomes 13 and X (in 0.3-1.3% of cells analyzed) [BenDavid et al, 2011;Nikitina et al, 2011], trisomies of chromosome 5 and, less frequently, of chromosomes 8 and 20 (in 15-20% of cell analyzed) [Grigorian et al, 2010;Tarte et al, 2010] were reported after conventional G-banding.…”

Section: Mesenchymal Stromal Stem Cellsmentioning

confidence: 99%

Chromosomal Abnormalities in Embryonic and Somatic Stem Cells

Rebuzzini¹,

Zuccotti

Redi³

et al. 2015

Cytogenet Genome Res

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The potential use of stem cells (SCs) for tissue engineering, regenerative medicine, disease modeling, toxicological studies, drug delivery, and as in vitro model for the study of basic developmental processes implies large-scale in vitro culture. Here, after a brief description of the main techniques used for karyotype analysis, we will give a detailed overview of the chromosome abnormalities described in pluripotent (embryonic and induced pluripotent SCs) and somatic SCs, and the possible causes of their origin during culture.

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“…However, as has been shown previously, the doubling time for cells cultured in the described conditions varies widely from 12-50 h for the first 43 doublings, and from 50-60 h after the 55th doubling. 29 Human MSCs were obtained from patients undergoing diagnostic biopsies after providing informed, written consent. Bone marrow blood was aspirated from the posterior iliac crest and the mononuclear fraction was isolated by gradient centrifugation.…”

Section: Cells and Culture Conditionsmentioning

confidence: 99%

Evaluation of Sericin as a Fetal Bovine Serum-Replacing Cryoprotectant During Freezing of Human Mesenchymal Stromal Cells and Human Osteoblast-Like Cells

Verdanova

Pytlik

Kalbáčová

2014

Biopreservation and Biobanking

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A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBSfree medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO.

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Mesenchymal Stem Cells Isolated from the Human Bone Marrow: Cultivation, Phenotypic Analysis and Changes in Proliferation Kinetics

Cited by 18 publications

References 16 publications

Human Dental Pulp Stem Cells – Isolation and Long Term Cultivation

Human Dental Pulp Stem Cells – Isolation and Long Term Cultivation

Chromosomal Abnormalities in Embryonic and Somatic Stem Cells

Evaluation of Sericin as a Fetal Bovine Serum-Replacing Cryoprotectant During Freezing of Human Mesenchymal Stromal Cells and Human Osteoblast-Like Cells

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