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Hematopoietic stem/progenitor cell sources to generate reticulocytes for Plasmodium vivax culture.

Noulin F, Manesia JK, Rosanas-Urgell A, Erhart A, Borlon C, Van Den Abbeele J, d'Alessandro U, Verfaillie CM - PLoS ONE (2014)

Bottom Line: We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB).Compared to CD34+-enriched populations of PB and BM, CD34+-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax.As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies.

View Article: PubMed Central - PubMed

Affiliation: Unit of Malariology, Institute of Tropical Medicine, Antwerp, Belgium.

ABSTRACT
The predilection of Plasmodium vivax (P. vivax) for reticulocytes is a major obstacle for its establishment in a long-term culture system, as this requires a continuous supply of large quantities of reticulocytes, representing only 1-2% of circulating red blood cells. We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB). Compared to CD34+-enriched populations of PB and BM, CD34+-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax. In addition, when CD34+-enriched cells were first expanded, a further extensive increase in reticulocytes was seen for UCB, to a lesser degree BM but not PB. As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies.

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Parasite densities 24 hours post-invasion with P. vivax.The parasite density was counted for at least 500 red blood cells, dividing the number of infected enucleated cells by the total number of cells and multiplied by 100 (%). a) Parasite density for different sources of HSPC-derived reticulocytes with 1 P. vivax isolate. The mean and SD of 2 different batches of differentiated reticulocytes was calculated for each source of HSPC and tested for invasion with the same P. vivax isolate. b) Parasite density for each source of reticulocytes with different P. vivax isolates. Parasite densities (%) were counted by dividing the number of P. vivax ring-infected cells by the total number of counted RBCs and multiplying the result by 100. Different reticulocyte sources were tested: grey  =  UCB/HSPC-derived reticulocytes; dotted  =  BM/HSPC-derived reticulocytes; squared  =  PBMC/HSPC-derived reticulocytes; white  =  reticulocytes concentrated from UCB, black  =  reticulocytes concentrated from adult peripheral blood. PV1 and PV2 were tested with the same batches of HSPC-derived reticulocytes for the 3 different sources (UCB, BM and PBMC). For PV4, the proportion of reticulocytes was 20% for HSCP-derived reticulocytes and respectively 70% and 60% for reticulocytes concentrated from UCB adult peripheral blood.
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pone-0112496-g004: Parasite densities 24 hours post-invasion with P. vivax.The parasite density was counted for at least 500 red blood cells, dividing the number of infected enucleated cells by the total number of cells and multiplied by 100 (%). a) Parasite density for different sources of HSPC-derived reticulocytes with 1 P. vivax isolate. The mean and SD of 2 different batches of differentiated reticulocytes was calculated for each source of HSPC and tested for invasion with the same P. vivax isolate. b) Parasite density for each source of reticulocytes with different P. vivax isolates. Parasite densities (%) were counted by dividing the number of P. vivax ring-infected cells by the total number of counted RBCs and multiplying the result by 100. Different reticulocyte sources were tested: grey  =  UCB/HSPC-derived reticulocytes; dotted  =  BM/HSPC-derived reticulocytes; squared  =  PBMC/HSPC-derived reticulocytes; white  =  reticulocytes concentrated from UCB, black  =  reticulocytes concentrated from adult peripheral blood. PV1 and PV2 were tested with the same batches of HSPC-derived reticulocytes for the 3 different sources (UCB, BM and PBMC). For PV4, the proportion of reticulocytes was 20% for HSCP-derived reticulocytes and respectively 70% and 60% for reticulocytes concentrated from UCB adult peripheral blood.

Mentions: When using the same P. vivax isolate, the invasion rate between different HSPC sources did not differ for all of the 4 P. vivax isolates tested (Figure 4a); however, the invasion rate observed varied for each of the P. vivax isolates used. When we compared two different P. vivax isolates using the same HSPC-derived reticulocytes, the invasion rate varied significantly by isolate (Figure 4b; PV1 and PV2, p<0.001). After 3 days of culture, only few rings (parasite density <0.05%) could be observed and none survived longer than 72 hours, regardless of the HSPC source. Interestingly, the invasion rate of P. vivax in HSPC-derived reticulocytes appeared to be higher when compared with reticulocytes isolated directly from PB. For the same P. vivax isolate, the parasite density 24 hours post-invasion in UCB/HSPC-derived reticulocytes was up to 9-fold higher than in UCB-concentrated reticulocytes (1.8% versus 0.2%, respectively), and 18-fold higher than adult PB-concentrated reticulocytes (2.1% versus 0.1% respectively). Parasite densities were not significantly different between HSPC-derived reticulocytes (5%), UCB-concentrated reticulocytes (4.6% p = 0.056) and PB-concentrated reticulocytes (3.5% p = 0.06) when the reticulocyte percentage was 20% for HSPC-derived reticulocytes and respectively 60% and 70% for reticulocytes concentrated from PB and UCB.


Hematopoietic stem/progenitor cell sources to generate reticulocytes for Plasmodium vivax culture.

Noulin F, Manesia JK, Rosanas-Urgell A, Erhart A, Borlon C, Van Den Abbeele J, d'Alessandro U, Verfaillie CM - PLoS ONE (2014)

Parasite densities 24 hours post-invasion with P. vivax.The parasite density was counted for at least 500 red blood cells, dividing the number of infected enucleated cells by the total number of cells and multiplied by 100 (%). a) Parasite density for different sources of HSPC-derived reticulocytes with 1 P. vivax isolate. The mean and SD of 2 different batches of differentiated reticulocytes was calculated for each source of HSPC and tested for invasion with the same P. vivax isolate. b) Parasite density for each source of reticulocytes with different P. vivax isolates. Parasite densities (%) were counted by dividing the number of P. vivax ring-infected cells by the total number of counted RBCs and multiplying the result by 100. Different reticulocyte sources were tested: grey  =  UCB/HSPC-derived reticulocytes; dotted  =  BM/HSPC-derived reticulocytes; squared  =  PBMC/HSPC-derived reticulocytes; white  =  reticulocytes concentrated from UCB, black  =  reticulocytes concentrated from adult peripheral blood. PV1 and PV2 were tested with the same batches of HSPC-derived reticulocytes for the 3 different sources (UCB, BM and PBMC). For PV4, the proportion of reticulocytes was 20% for HSCP-derived reticulocytes and respectively 70% and 60% for reticulocytes concentrated from UCB adult peripheral blood.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4231068&req=5

pone-0112496-g004: Parasite densities 24 hours post-invasion with P. vivax.The parasite density was counted for at least 500 red blood cells, dividing the number of infected enucleated cells by the total number of cells and multiplied by 100 (%). a) Parasite density for different sources of HSPC-derived reticulocytes with 1 P. vivax isolate. The mean and SD of 2 different batches of differentiated reticulocytes was calculated for each source of HSPC and tested for invasion with the same P. vivax isolate. b) Parasite density for each source of reticulocytes with different P. vivax isolates. Parasite densities (%) were counted by dividing the number of P. vivax ring-infected cells by the total number of counted RBCs and multiplying the result by 100. Different reticulocyte sources were tested: grey  =  UCB/HSPC-derived reticulocytes; dotted  =  BM/HSPC-derived reticulocytes; squared  =  PBMC/HSPC-derived reticulocytes; white  =  reticulocytes concentrated from UCB, black  =  reticulocytes concentrated from adult peripheral blood. PV1 and PV2 were tested with the same batches of HSPC-derived reticulocytes for the 3 different sources (UCB, BM and PBMC). For PV4, the proportion of reticulocytes was 20% for HSCP-derived reticulocytes and respectively 70% and 60% for reticulocytes concentrated from UCB adult peripheral blood.
Mentions: When using the same P. vivax isolate, the invasion rate between different HSPC sources did not differ for all of the 4 P. vivax isolates tested (Figure 4a); however, the invasion rate observed varied for each of the P. vivax isolates used. When we compared two different P. vivax isolates using the same HSPC-derived reticulocytes, the invasion rate varied significantly by isolate (Figure 4b; PV1 and PV2, p<0.001). After 3 days of culture, only few rings (parasite density <0.05%) could be observed and none survived longer than 72 hours, regardless of the HSPC source. Interestingly, the invasion rate of P. vivax in HSPC-derived reticulocytes appeared to be higher when compared with reticulocytes isolated directly from PB. For the same P. vivax isolate, the parasite density 24 hours post-invasion in UCB/HSPC-derived reticulocytes was up to 9-fold higher than in UCB-concentrated reticulocytes (1.8% versus 0.2%, respectively), and 18-fold higher than adult PB-concentrated reticulocytes (2.1% versus 0.1% respectively). Parasite densities were not significantly different between HSPC-derived reticulocytes (5%), UCB-concentrated reticulocytes (4.6% p = 0.056) and PB-concentrated reticulocytes (3.5% p = 0.06) when the reticulocyte percentage was 20% for HSPC-derived reticulocytes and respectively 60% and 70% for reticulocytes concentrated from PB and UCB.

Bottom Line: We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB).Compared to CD34+-enriched populations of PB and BM, CD34+-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax.As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies.

View Article: PubMed Central - PubMed

Affiliation: Unit of Malariology, Institute of Tropical Medicine, Antwerp, Belgium.

ABSTRACT
The predilection of Plasmodium vivax (P. vivax) for reticulocytes is a major obstacle for its establishment in a long-term culture system, as this requires a continuous supply of large quantities of reticulocytes, representing only 1-2% of circulating red blood cells. We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB). Compared to CD34+-enriched populations of PB and BM, CD34+-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax. In addition, when CD34+-enriched cells were first expanded, a further extensive increase in reticulocytes was seen for UCB, to a lesser degree BM but not PB. As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies.

Show MeSH
Related in: MedlinePlus