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Differentiation of human umbilical cord matrix mesenchymal stem cells into neural-like progenitor cells and maturation into an oligodendroglial-like lineage.

Leite C, Silva NT, Mendes S, Ribeiro A, de Faria JP, Lourenço T, dos Santos F, Andrade PZ, Cardoso CM, Vieira M, Paiva A, da Silva CL, Cabral JM, Relvas JB, Grãos M - PLoS ONE (2014)

Bottom Line: In the present work MSCs were isolated from the umbilical cord matrix (Wharton's jelly) of human umbilical cord samples.The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity.The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of bona-fide oligodendrocyte precursor cells and differentiated oligodendrocytes.

View Article: PubMed Central - PubMed

Affiliation: Biocant - Technology Transfer Association, Biocant Park, Cantanhede, Portugal.

ABSTRACT
Mesenchymal stem cells (MSCs) are viewed as safe, readily available and promising adult stem cells, which are currently used in several clinical trials. Additionally, their soluble-factor secretion and multi-lineage differentiation capacities place MSCs in the forefront of stem cell types with expected near-future clinical applications. In the present work MSCs were isolated from the umbilical cord matrix (Wharton's jelly) of human umbilical cord samples. The cells were thoroughly characterized and confirmed as bona-fide MSCs, presenting in vitro low generation time, high proliferative and colony-forming unit-fibroblast (CFU-F) capacity, typical MSC immunophenotype and osteogenic, chondrogenic and adipogenic differentiation capacity. The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity. The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of bona-fide oligodendrocyte precursor cells and differentiated oligodendrocytes.

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Proliferation and colony-forming capacity of human umbilical cord mesenchymal stem cells (hUCM-MSCs) in vitro.Total number of cells (hUCM-MSCs) along time (days) and passage number (A, B), generation time (C), population doubling (D), cumulative population doubling (E) from passage 1 (P1) or P2 until P8, as indicated, and colony forming unit-fibroblast (CFU-F) number at P2 and P8 (F). Cells were isolated from 3 distinct human umbilical cord matrix samples (CM#2, #3 and #7). From P2 onwards (inclusively), cells were plated at a fixed density of 3,000 cells/cm2, allowed to proliferate until sub-confluence and re-plated in the same way (A-E), or plated at 3 cells/cm2 at P2 and P8 and cultured for 15 days for the CFU-F study (F). The total number of cells (TNC) was determined at each passage (P1-P8) by cumulative counting of the cells once they reached a confluence of 80% (A, B). The TNC designates the theoretical number of cells that could be obtained if no cells were discarded between each passage. The observed mean generation time (GT) was between 1.02 (±0.071) and 1.55 (±0.316) days (C), and no statistically significant differences were found in GT from passages 2 to 8 (Kruskal-Wallis one-way ANOVA followed by Dunn's Multiple Comparison test). The CFU-F capacity was maintained from P2 to P8 (F) and no statistically significant differences were found (two-tailed Mann-Whitney test). Bars represent mean ± SEM (B-F).
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pone-0111059-g001: Proliferation and colony-forming capacity of human umbilical cord mesenchymal stem cells (hUCM-MSCs) in vitro.Total number of cells (hUCM-MSCs) along time (days) and passage number (A, B), generation time (C), population doubling (D), cumulative population doubling (E) from passage 1 (P1) or P2 until P8, as indicated, and colony forming unit-fibroblast (CFU-F) number at P2 and P8 (F). Cells were isolated from 3 distinct human umbilical cord matrix samples (CM#2, #3 and #7). From P2 onwards (inclusively), cells were plated at a fixed density of 3,000 cells/cm2, allowed to proliferate until sub-confluence and re-plated in the same way (A-E), or plated at 3 cells/cm2 at P2 and P8 and cultured for 15 days for the CFU-F study (F). The total number of cells (TNC) was determined at each passage (P1-P8) by cumulative counting of the cells once they reached a confluence of 80% (A, B). The TNC designates the theoretical number of cells that could be obtained if no cells were discarded between each passage. The observed mean generation time (GT) was between 1.02 (±0.071) and 1.55 (±0.316) days (C), and no statistically significant differences were found in GT from passages 2 to 8 (Kruskal-Wallis one-way ANOVA followed by Dunn's Multiple Comparison test). The CFU-F capacity was maintained from P2 to P8 (F) and no statistically significant differences were found (two-tailed Mann-Whitney test). Bars represent mean ± SEM (B-F).

Mentions: UCM-MSCs have been described to proliferate readily in vitro[34], [35], hence we sought to thoroughly characterize their proliferation kinetics, namely the total number of cells - TNC (Figures 1A, B), generation time - GT (Figure 1C), population doubling - PD (Figure 1D) and cumulative population doubling - CPD (Figure 1E) of cells isolated from 3 randomly selected independent samples (CM#2, CM#3 and CM#7) between passages 2 and 8 (P2-P8). Notably, by the end of P4 (17 to 21 days after the initial isolation of the UCM explants), the total number of cells obtained from each sample (i.e., if no cells had been discarded until that point) had surpassed 1×109 cells (Figures 1A, B), well above what is considered relevant for clinical applications [29] (about 1–2 million cells per kg of body weight) and at passage 8 the TNC was 5.98×1013 (±4.76×1013). On average, between P2 and P8, the generation time ranged from 1.02±0.071 (SEM) to 1.55±0.316 (SEM) days (Figure 1C). Moreover, the colony-forming unit-fibroblast (CFU-F) capacity of the cells (Figure 1F) was maintained throughout P2 to P8 [48.5±7.52 (SEM) and 43.8±16.20 (SEM)]. For both types of assays (GT and CFU-F), no statistically significant differences were found between the time points, indicating that neither the generation time nor the CFU-F capacity were significantly affected by passaging the cells until P8. Hence, the cells showed high proliferative capacity typical of bona-fide MSCs, maintained a short generation time from passages 2 to 8 and reached a clinically relevant number (superior to 1×109 cells) within a time-frame of 17 to 21 days.


Differentiation of human umbilical cord matrix mesenchymal stem cells into neural-like progenitor cells and maturation into an oligodendroglial-like lineage.

Leite C, Silva NT, Mendes S, Ribeiro A, de Faria JP, Lourenço T, dos Santos F, Andrade PZ, Cardoso CM, Vieira M, Paiva A, da Silva CL, Cabral JM, Relvas JB, Grãos M - PLoS ONE (2014)

Proliferation and colony-forming capacity of human umbilical cord mesenchymal stem cells (hUCM-MSCs) in vitro.Total number of cells (hUCM-MSCs) along time (days) and passage number (A, B), generation time (C), population doubling (D), cumulative population doubling (E) from passage 1 (P1) or P2 until P8, as indicated, and colony forming unit-fibroblast (CFU-F) number at P2 and P8 (F). Cells were isolated from 3 distinct human umbilical cord matrix samples (CM#2, #3 and #7). From P2 onwards (inclusively), cells were plated at a fixed density of 3,000 cells/cm2, allowed to proliferate until sub-confluence and re-plated in the same way (A-E), or plated at 3 cells/cm2 at P2 and P8 and cultured for 15 days for the CFU-F study (F). The total number of cells (TNC) was determined at each passage (P1-P8) by cumulative counting of the cells once they reached a confluence of 80% (A, B). The TNC designates the theoretical number of cells that could be obtained if no cells were discarded between each passage. The observed mean generation time (GT) was between 1.02 (±0.071) and 1.55 (±0.316) days (C), and no statistically significant differences were found in GT from passages 2 to 8 (Kruskal-Wallis one-way ANOVA followed by Dunn's Multiple Comparison test). The CFU-F capacity was maintained from P2 to P8 (F) and no statistically significant differences were found (two-tailed Mann-Whitney test). Bars represent mean ± SEM (B-F).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214693&req=5

pone-0111059-g001: Proliferation and colony-forming capacity of human umbilical cord mesenchymal stem cells (hUCM-MSCs) in vitro.Total number of cells (hUCM-MSCs) along time (days) and passage number (A, B), generation time (C), population doubling (D), cumulative population doubling (E) from passage 1 (P1) or P2 until P8, as indicated, and colony forming unit-fibroblast (CFU-F) number at P2 and P8 (F). Cells were isolated from 3 distinct human umbilical cord matrix samples (CM#2, #3 and #7). From P2 onwards (inclusively), cells were plated at a fixed density of 3,000 cells/cm2, allowed to proliferate until sub-confluence and re-plated in the same way (A-E), or plated at 3 cells/cm2 at P2 and P8 and cultured for 15 days for the CFU-F study (F). The total number of cells (TNC) was determined at each passage (P1-P8) by cumulative counting of the cells once they reached a confluence of 80% (A, B). The TNC designates the theoretical number of cells that could be obtained if no cells were discarded between each passage. The observed mean generation time (GT) was between 1.02 (±0.071) and 1.55 (±0.316) days (C), and no statistically significant differences were found in GT from passages 2 to 8 (Kruskal-Wallis one-way ANOVA followed by Dunn's Multiple Comparison test). The CFU-F capacity was maintained from P2 to P8 (F) and no statistically significant differences were found (two-tailed Mann-Whitney test). Bars represent mean ± SEM (B-F).
Mentions: UCM-MSCs have been described to proliferate readily in vitro[34], [35], hence we sought to thoroughly characterize their proliferation kinetics, namely the total number of cells - TNC (Figures 1A, B), generation time - GT (Figure 1C), population doubling - PD (Figure 1D) and cumulative population doubling - CPD (Figure 1E) of cells isolated from 3 randomly selected independent samples (CM#2, CM#3 and CM#7) between passages 2 and 8 (P2-P8). Notably, by the end of P4 (17 to 21 days after the initial isolation of the UCM explants), the total number of cells obtained from each sample (i.e., if no cells had been discarded until that point) had surpassed 1×109 cells (Figures 1A, B), well above what is considered relevant for clinical applications [29] (about 1–2 million cells per kg of body weight) and at passage 8 the TNC was 5.98×1013 (±4.76×1013). On average, between P2 and P8, the generation time ranged from 1.02±0.071 (SEM) to 1.55±0.316 (SEM) days (Figure 1C). Moreover, the colony-forming unit-fibroblast (CFU-F) capacity of the cells (Figure 1F) was maintained throughout P2 to P8 [48.5±7.52 (SEM) and 43.8±16.20 (SEM)]. For both types of assays (GT and CFU-F), no statistically significant differences were found between the time points, indicating that neither the generation time nor the CFU-F capacity were significantly affected by passaging the cells until P8. Hence, the cells showed high proliferative capacity typical of bona-fide MSCs, maintained a short generation time from passages 2 to 8 and reached a clinically relevant number (superior to 1×109 cells) within a time-frame of 17 to 21 days.

Bottom Line: In the present work MSCs were isolated from the umbilical cord matrix (Wharton's jelly) of human umbilical cord samples.The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity.The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of bona-fide oligodendrocyte precursor cells and differentiated oligodendrocytes.

View Article: PubMed Central - PubMed

Affiliation: Biocant - Technology Transfer Association, Biocant Park, Cantanhede, Portugal.

ABSTRACT
Mesenchymal stem cells (MSCs) are viewed as safe, readily available and promising adult stem cells, which are currently used in several clinical trials. Additionally, their soluble-factor secretion and multi-lineage differentiation capacities place MSCs in the forefront of stem cell types with expected near-future clinical applications. In the present work MSCs were isolated from the umbilical cord matrix (Wharton's jelly) of human umbilical cord samples. The cells were thoroughly characterized and confirmed as bona-fide MSCs, presenting in vitro low generation time, high proliferative and colony-forming unit-fibroblast (CFU-F) capacity, typical MSC immunophenotype and osteogenic, chondrogenic and adipogenic differentiation capacity. The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity. The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of bona-fide oligodendrocyte precursor cells and differentiated oligodendrocytes.

Show MeSH
Related in: MedlinePlus