Limits...
Mesenchymal stem cells as a feeder layer can prevent apoptosis of expanded hematopoietic stem cells derived from cord blood.

Mehrasa R, Vaziri H, Oodi A, Khorshidfar M, Nikogoftar M, Golpour M, Amirizadeh N - Int J Mol Cell Med (2014)

Bottom Line: Expansion was followed by measuring the total nucleated cells (TNCs), CD34(+) (‏) cells and colony-forming unit (CFU) output.Flow cytometry analysis of stained cells by annexin V and propidium iodide was performed for detection of apoptosis rate and cell cycle distribution in expanded cells.The expansion of cord blood HSCs on MSCs as a feeder layer resulted in higher proliferation and reduction in apoptosis rate.

View Article: PubMed Central - PubMed

Affiliation: University of Guilan, Rasht, Iran.

ABSTRACT
Umbilical cord blood (UCB) has been used for transplantation in the treatment of hematologic disorders as a source of hematopoietic stem cells (HSCs). Because of insufficient number of cord blood CD34(+) cells, the expansion of these cells seems to be important for clinical application. Mesenchymal stromal cells (MSCs), playing an important role in HSCs maintenance, were used as feeder layer. Apoptosis and cell cycle distribution of expanded cells were analyzed in MSCs co-culture and cytokine conditions and results were compared. Three culture conditions of cord blood HSCs were prepared ex-vivo for 14 days: cytokines (SCF, TPO and Flt3L) with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs without cytokines. Expansion was followed by measuring the total nucleated cells (TNCs), CD34(+) (‏) cells and colony-forming unit (CFU) output. Flow cytometry analysis of stained cells by annexin V and propidium iodide was performed for detection of apoptosis rate and cell cycle distribution in expanded cells. Maximum cord blood CD34(+) cells expansion was observed in day 10. The mean fold change of TNCs and CD34+ cells at day 10 in the co-culture system with cytokines was significantly higher than the cytokine culture without MSCs feeder layer and co-culture system without cytokines (n=6, p=0.023). The highest apoptosis rate and the least number of cells in Go/G1 phase were observed in cytokine culture without feeder layer (p=0.041). The expansion of cord blood HSCs on MSCs as a feeder layer resulted in higher proliferation and reduction in apoptosis rate.

No MeSH data available.


Related in: MedlinePlus

Flow cytometric analysis of the percentage of CD34+ expanded cells in different culture conditions. Flow cytometric analysis of fresh CD34+ enriched cells (A) and expanded cells in the co-culture system without cytokine (B) cytokine culture without MSCs (C) and with MSCs (D) at day 10 of expansion: Specific staining was performed with anti-CD34-FITC and anti-CD38-PE antibodies. FL1: CD34, FL2: CD38; A. CD34: 62.09 %, CD38: 23.87%; B. CD34: 70.07 %, CD38: 54.99%; c. CD34: 62.01 %, CD38: 56.84%; D. CD34: 78.75 %, CD38: 61.96%.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3927388&req=5

Figure 2: Flow cytometric analysis of the percentage of CD34+ expanded cells in different culture conditions. Flow cytometric analysis of fresh CD34+ enriched cells (A) and expanded cells in the co-culture system without cytokine (B) cytokine culture without MSCs (C) and with MSCs (D) at day 10 of expansion: Specific staining was performed with anti-CD34-FITC and anti-CD38-PE antibodies. FL1: CD34, FL2: CD38; A. CD34: 62.09 %, CD38: 23.87%; B. CD34: 70.07 %, CD38: 54.99%; c. CD34: 62.01 %, CD38: 56.84%; D. CD34: 78.75 %, CD38: 61.96%.

Mentions: The purity of separated CD34 + cells determi-ned by flow cytometry analysis, was 88% ± 12. Moreover, 31.45% ± 7 of them were positive for CD38 marker. Lineage specific CD markers expression was low (n=3) (Figure 2).


Mesenchymal stem cells as a feeder layer can prevent apoptosis of expanded hematopoietic stem cells derived from cord blood.

Mehrasa R, Vaziri H, Oodi A, Khorshidfar M, Nikogoftar M, Golpour M, Amirizadeh N - Int J Mol Cell Med (2014)

Flow cytometric analysis of the percentage of CD34+ expanded cells in different culture conditions. Flow cytometric analysis of fresh CD34+ enriched cells (A) and expanded cells in the co-culture system without cytokine (B) cytokine culture without MSCs (C) and with MSCs (D) at day 10 of expansion: Specific staining was performed with anti-CD34-FITC and anti-CD38-PE antibodies. FL1: CD34, FL2: CD38; A. CD34: 62.09 %, CD38: 23.87%; B. CD34: 70.07 %, CD38: 54.99%; c. CD34: 62.01 %, CD38: 56.84%; D. CD34: 78.75 %, CD38: 61.96%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927388&req=5

Figure 2: Flow cytometric analysis of the percentage of CD34+ expanded cells in different culture conditions. Flow cytometric analysis of fresh CD34+ enriched cells (A) and expanded cells in the co-culture system without cytokine (B) cytokine culture without MSCs (C) and with MSCs (D) at day 10 of expansion: Specific staining was performed with anti-CD34-FITC and anti-CD38-PE antibodies. FL1: CD34, FL2: CD38; A. CD34: 62.09 %, CD38: 23.87%; B. CD34: 70.07 %, CD38: 54.99%; c. CD34: 62.01 %, CD38: 56.84%; D. CD34: 78.75 %, CD38: 61.96%.
Mentions: The purity of separated CD34 + cells determi-ned by flow cytometry analysis, was 88% ± 12. Moreover, 31.45% ± 7 of them were positive for CD38 marker. Lineage specific CD markers expression was low (n=3) (Figure 2).

Bottom Line: Expansion was followed by measuring the total nucleated cells (TNCs), CD34(+) (‏) cells and colony-forming unit (CFU) output.Flow cytometry analysis of stained cells by annexin V and propidium iodide was performed for detection of apoptosis rate and cell cycle distribution in expanded cells.The expansion of cord blood HSCs on MSCs as a feeder layer resulted in higher proliferation and reduction in apoptosis rate.

View Article: PubMed Central - PubMed

Affiliation: University of Guilan, Rasht, Iran.

ABSTRACT
Umbilical cord blood (UCB) has been used for transplantation in the treatment of hematologic disorders as a source of hematopoietic stem cells (HSCs). Because of insufficient number of cord blood CD34(+) cells, the expansion of these cells seems to be important for clinical application. Mesenchymal stromal cells (MSCs), playing an important role in HSCs maintenance, were used as feeder layer. Apoptosis and cell cycle distribution of expanded cells were analyzed in MSCs co-culture and cytokine conditions and results were compared. Three culture conditions of cord blood HSCs were prepared ex-vivo for 14 days: cytokines (SCF, TPO and Flt3L) with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs without cytokines. Expansion was followed by measuring the total nucleated cells (TNCs), CD34(+) (‏) cells and colony-forming unit (CFU) output. Flow cytometry analysis of stained cells by annexin V and propidium iodide was performed for detection of apoptosis rate and cell cycle distribution in expanded cells. Maximum cord blood CD34(+) cells expansion was observed in day 10. The mean fold change of TNCs and CD34+ cells at day 10 in the co-culture system with cytokines was significantly higher than the cytokine culture without MSCs feeder layer and co-culture system without cytokines (n=6, p=0.023). The highest apoptosis rate and the least number of cells in Go/G1 phase were observed in cytokine culture without feeder layer (p=0.041). The expansion of cord blood HSCs on MSCs as a feeder layer resulted in higher proliferation and reduction in apoptosis rate.

No MeSH data available.


Related in: MedlinePlus