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High log-scale expansion of functional human natural killer cells from umbilical cord blood CD34-positive cells for adoptive cancer immunotherapy.

Spanholtz J, Tordoir M, Eissens D, Preijers F, van der Meer A, Joosten I, Schaap N, de Witte TM, Dolstra H - PLoS ONE (2010)

Bottom Line: Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol.Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors.Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hematology, Department of Laboratory Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT
Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56(+)CD3(-) NK cell products could be routinely generated from freshly selected CD34(+) UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34(+) UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

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Ex vivo generation of CD56+ NK cells from cytokine-expanded CD34+ UCB cells.CD34-enriched UCB cells were expanded for two weeks using three different media (H3000, Stemline I and Stemline II) and subsequently differentiated into NK cells for three additional weeks in the same basal medium using Method I (see Figure 1). Cell cultures were weekly analyzed for cell numbers and phenotype using FCM. (a) Representative example of antigen expression during the two-step culture period using H3000 medium. One week after the onset of the NK cell differentiation step 2 (i.e. after 3 weeks total culture duration) the CD56+CD161+CD94+ NK cell population increases and reaches high purity after 3 weeks of differentiation (i.e week 5). (b) Mean CD56+ cell frequency during the 5 week culture period for three different media, which have been tested in parallel experiments using 3–6 UCB donors. (c) Mean total CD56+ NK cell numbers after initial seeding of 1×104 CD34+ UCB cells during 5 weeks of culture using Method I. Data represent a theoretical calculation based on the actual expansion rates of CD56+ cells. Total yield of CD56+ cells at each week was calculated by multiplying the expansion rate per week with the number of cultured cells. (d) Mean fold expansion of total cells after initial seeding of 1×104 CD34+ UCB cells during 5 weeks of culture using Method I. Data represent a theoretical calculation based on the actual expansion rates of total cells.
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pone-0009221-g002: Ex vivo generation of CD56+ NK cells from cytokine-expanded CD34+ UCB cells.CD34-enriched UCB cells were expanded for two weeks using three different media (H3000, Stemline I and Stemline II) and subsequently differentiated into NK cells for three additional weeks in the same basal medium using Method I (see Figure 1). Cell cultures were weekly analyzed for cell numbers and phenotype using FCM. (a) Representative example of antigen expression during the two-step culture period using H3000 medium. One week after the onset of the NK cell differentiation step 2 (i.e. after 3 weeks total culture duration) the CD56+CD161+CD94+ NK cell population increases and reaches high purity after 3 weeks of differentiation (i.e week 5). (b) Mean CD56+ cell frequency during the 5 week culture period for three different media, which have been tested in parallel experiments using 3–6 UCB donors. (c) Mean total CD56+ NK cell numbers after initial seeding of 1×104 CD34+ UCB cells during 5 weeks of culture using Method I. Data represent a theoretical calculation based on the actual expansion rates of CD56+ cells. Total yield of CD56+ cells at each week was calculated by multiplying the expansion rate per week with the number of cultured cells. (d) Mean fold expansion of total cells after initial seeding of 1×104 CD34+ UCB cells during 5 weeks of culture using Method I. Data represent a theoretical calculation based on the actual expansion rates of total cells.

Mentions: Next, we investigated whether the expanded UCB-derived CD34+ cells were able to differentiate into CD56+ NK cells. The differentiation step was monitored by the analysis of the cell surface molecules CD34, CD117, CD56, CD94 and CD161, which have been described to be expressed at different human NK cell developmental stages in vivo [18]. We observed after 3 weeks total culture duration that the percentage of CD34+ cells further declined, while the CD56+CD161+CD94+ NK cell population increased to 10–18% (e.g. Figure 2a). Thereafter, the population of CD56+CD161+CD94+ cells rapidly increased to 60–77% after 4 weeks and 80–96% after 5 weeks of culture (e.g. Figure 2a).


High log-scale expansion of functional human natural killer cells from umbilical cord blood CD34-positive cells for adoptive cancer immunotherapy.

Spanholtz J, Tordoir M, Eissens D, Preijers F, van der Meer A, Joosten I, Schaap N, de Witte TM, Dolstra H - PLoS ONE (2010)

Ex vivo generation of CD56+ NK cells from cytokine-expanded CD34+ UCB cells.CD34-enriched UCB cells were expanded for two weeks using three different media (H3000, Stemline I and Stemline II) and subsequently differentiated into NK cells for three additional weeks in the same basal medium using Method I (see Figure 1). Cell cultures were weekly analyzed for cell numbers and phenotype using FCM. (a) Representative example of antigen expression during the two-step culture period using H3000 medium. One week after the onset of the NK cell differentiation step 2 (i.e. after 3 weeks total culture duration) the CD56+CD161+CD94+ NK cell population increases and reaches high purity after 3 weeks of differentiation (i.e week 5). (b) Mean CD56+ cell frequency during the 5 week culture period for three different media, which have been tested in parallel experiments using 3–6 UCB donors. (c) Mean total CD56+ NK cell numbers after initial seeding of 1×104 CD34+ UCB cells during 5 weeks of culture using Method I. Data represent a theoretical calculation based on the actual expansion rates of CD56+ cells. Total yield of CD56+ cells at each week was calculated by multiplying the expansion rate per week with the number of cultured cells. (d) Mean fold expansion of total cells after initial seeding of 1×104 CD34+ UCB cells during 5 weeks of culture using Method I. Data represent a theoretical calculation based on the actual expansion rates of total cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2821405&req=5

pone-0009221-g002: Ex vivo generation of CD56+ NK cells from cytokine-expanded CD34+ UCB cells.CD34-enriched UCB cells were expanded for two weeks using three different media (H3000, Stemline I and Stemline II) and subsequently differentiated into NK cells for three additional weeks in the same basal medium using Method I (see Figure 1). Cell cultures were weekly analyzed for cell numbers and phenotype using FCM. (a) Representative example of antigen expression during the two-step culture period using H3000 medium. One week after the onset of the NK cell differentiation step 2 (i.e. after 3 weeks total culture duration) the CD56+CD161+CD94+ NK cell population increases and reaches high purity after 3 weeks of differentiation (i.e week 5). (b) Mean CD56+ cell frequency during the 5 week culture period for three different media, which have been tested in parallel experiments using 3–6 UCB donors. (c) Mean total CD56+ NK cell numbers after initial seeding of 1×104 CD34+ UCB cells during 5 weeks of culture using Method I. Data represent a theoretical calculation based on the actual expansion rates of CD56+ cells. Total yield of CD56+ cells at each week was calculated by multiplying the expansion rate per week with the number of cultured cells. (d) Mean fold expansion of total cells after initial seeding of 1×104 CD34+ UCB cells during 5 weeks of culture using Method I. Data represent a theoretical calculation based on the actual expansion rates of total cells.
Mentions: Next, we investigated whether the expanded UCB-derived CD34+ cells were able to differentiate into CD56+ NK cells. The differentiation step was monitored by the analysis of the cell surface molecules CD34, CD117, CD56, CD94 and CD161, which have been described to be expressed at different human NK cell developmental stages in vivo [18]. We observed after 3 weeks total culture duration that the percentage of CD34+ cells further declined, while the CD56+CD161+CD94+ NK cell population increased to 10–18% (e.g. Figure 2a). Thereafter, the population of CD56+CD161+CD94+ cells rapidly increased to 60–77% after 4 weeks and 80–96% after 5 weeks of culture (e.g. Figure 2a).

Bottom Line: Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol.Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors.Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hematology, Department of Laboratory Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT
Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56(+)CD3(-) NK cell products could be routinely generated from freshly selected CD34(+) UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34(+) UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56(+) NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34(+) cells for cancer immunotherapy.

Show MeSH
Related in: MedlinePlus