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Sustained Engraftment of Cryopreserved Human Bone Marrow CD34(+) Cells in Young Adult NSG Mice.

Wiekmeijer AS, Pike-Overzet K, Brugman MH, Salvatori DC, Egeler RM, Bredius RG, Fibbe WE, Staal FJ - Biores Open Access (2014)

Bottom Line: This protocol results in robust and reproducible high levels of lympho-myeloid engraftment.Similar results were obtained with cryopreserved human bone marrow samples, thus circumventing the need for fresh cells and allowing the use of patient derived bio-bank samples.Our findings have implications for use of this model in fundamental stem cell research, immunological studies in vivo and preclinical evaluations for HSC transplantation, expansion, and genetic modification.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center , Leiden, The Netherlands .

ABSTRACT
Hematopoietic stem cells (HSCs) are defined by their ability to repopulate the bone marrow of myeloablative conditioned and/or (lethally) irradiated recipients. To study the repopulating potential of human HSCs, murine models have been developed that rely on the use of immunodeficient mice that allow engraftment of human cells. The NSG xenograft model has emerged as the current standard for this purpose allowing for engraftment and study of human T cells. Here, we describe adaptations to the original NSG xenograft model that can be readily implemented. These adaptations encompass use of adult mice instead of newborns and a short ex vivo culture. This protocol results in robust and reproducible high levels of lympho-myeloid engraftment. Immunization of recipient mice with relevant antigen resulted in specific antibody formation, showing that both T cells and B cells were functional. In addition, bone marrow cells from primary recipients exhibited repopulating ability following transplantation into secondary recipients. Similar results were obtained with cryopreserved human bone marrow samples, thus circumventing the need for fresh cells and allowing the use of patient derived bio-bank samples. Our findings have implications for use of this model in fundamental stem cell research, immunological studies in vivo and preclinical evaluations for HSC transplantation, expansion, and genetic modification.

No MeSH data available.


Related in: MedlinePlus

Engraftment capacity of hematopoietic stem and progenitor cells (HSPCs) is not affected by a short in vitro culture. (A) Phenotype of transplanted cells. Plots are gated on the population indicated on top. (B) Engraftment as measured by percentage of human CD45 expressing cells in peripheral blood of humanized NSG mice transplanted with different cell doses of either fresh or cultured HSPCs isolated from umbilical cord blood (UCB). (C) Different cell lineages in peripheral blood of mice transplanted with 1.5×105 noncultured HSPCs isolated from UCB (top) or 1.5×105 cultured HSPCs (bottom). Myeloid cells (left), B cells (middle), and T cells (right) were gated within human CD45+ cells. (D) Contribution of different cell lineages in peripheral blood at 18 weeks after transplantation. Percentages are gated within human CD45+ cells. (E) Numbers of long-term-HSC (hCD45+CD34+CD38−CD90+CD45RA−CD49f+) present in bone marrow (BM) of NSG recipients transplanted with different cell doses of either fresh or cultured HSPCs isolated from UCB. Data are represented as mean±standard deviation (SD) in (B, D, E). 50k, 0.5×105 cells; 150k, 1.5×105cells. HSPCs were isolated from a pool of seven UCB donors, three mice per group.
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f1: Engraftment capacity of hematopoietic stem and progenitor cells (HSPCs) is not affected by a short in vitro culture. (A) Phenotype of transplanted cells. Plots are gated on the population indicated on top. (B) Engraftment as measured by percentage of human CD45 expressing cells in peripheral blood of humanized NSG mice transplanted with different cell doses of either fresh or cultured HSPCs isolated from umbilical cord blood (UCB). (C) Different cell lineages in peripheral blood of mice transplanted with 1.5×105 noncultured HSPCs isolated from UCB (top) or 1.5×105 cultured HSPCs (bottom). Myeloid cells (left), B cells (middle), and T cells (right) were gated within human CD45+ cells. (D) Contribution of different cell lineages in peripheral blood at 18 weeks after transplantation. Percentages are gated within human CD45+ cells. (E) Numbers of long-term-HSC (hCD45+CD34+CD38−CD90+CD45RA−CD49f+) present in bone marrow (BM) of NSG recipients transplanted with different cell doses of either fresh or cultured HSPCs isolated from UCB. Data are represented as mean±standard deviation (SD) in (B, D, E). 50k, 0.5×105 cells; 150k, 1.5×105cells. HSPCs were isolated from a pool of seven UCB donors, three mice per group.

Mentions: For genetic modification (e.g., gene therapy) HSPCs need to be cultured for a short period. To test whether this is feasible for the NSG model, we cultured HSPCs isolated from UCB overnight to determine the effect on engraftment potential. NSG mice were transplanted with 0.5×105 or 1.5×105 HSPCs directly after isolation or after overnight culture in StemSpan supplemented with cytokines. The transplanted cells were analyzed for expression of surface markers that define human HSC subsets.14 An increase in the percentage of CD90+CD45RA− cells and, within this population, CD49f+ cells was observed after overnight culture (Fig. 1A). This increase of more primitive HSC subsets was associated with similar engraftment of human cells in lymphoid organs (Fig. 1B) and similar contribution of cell lineages to engraftment in peripheral blood between recipients of fresh or cultured HSPCs (Fig. 1C and 1D). Engraftment of human cells and lineage contribution were both unaffected by the number of transplanted cells. No differences in numbers of cells belonging to the most primitive human HSC subset were found in the BM of recipients of fresh and cultured HSPCs, although there was a trend at lower transplanted cell numbers (0.5×105, 50,000) for more primitive stem cells to be detected in the BM of recipients of cultured HSPCs (Fig. 1E). This led us to conclude that the short overnight culture, as needed for genetic modification, does not compromise engraftment potential and lineage contribution of human HSPCs in primary recipients. Furthermore, the described protocol allows for a high level of engraftment in different organs (Fig. 1B).


Sustained Engraftment of Cryopreserved Human Bone Marrow CD34(+) Cells in Young Adult NSG Mice.

Wiekmeijer AS, Pike-Overzet K, Brugman MH, Salvatori DC, Egeler RM, Bredius RG, Fibbe WE, Staal FJ - Biores Open Access (2014)

Engraftment capacity of hematopoietic stem and progenitor cells (HSPCs) is not affected by a short in vitro culture. (A) Phenotype of transplanted cells. Plots are gated on the population indicated on top. (B) Engraftment as measured by percentage of human CD45 expressing cells in peripheral blood of humanized NSG mice transplanted with different cell doses of either fresh or cultured HSPCs isolated from umbilical cord blood (UCB). (C) Different cell lineages in peripheral blood of mice transplanted with 1.5×105 noncultured HSPCs isolated from UCB (top) or 1.5×105 cultured HSPCs (bottom). Myeloid cells (left), B cells (middle), and T cells (right) were gated within human CD45+ cells. (D) Contribution of different cell lineages in peripheral blood at 18 weeks after transplantation. Percentages are gated within human CD45+ cells. (E) Numbers of long-term-HSC (hCD45+CD34+CD38−CD90+CD45RA−CD49f+) present in bone marrow (BM) of NSG recipients transplanted with different cell doses of either fresh or cultured HSPCs isolated from UCB. Data are represented as mean±standard deviation (SD) in (B, D, E). 50k, 0.5×105 cells; 150k, 1.5×105cells. HSPCs were isolated from a pool of seven UCB donors, three mice per group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048975&req=5

f1: Engraftment capacity of hematopoietic stem and progenitor cells (HSPCs) is not affected by a short in vitro culture. (A) Phenotype of transplanted cells. Plots are gated on the population indicated on top. (B) Engraftment as measured by percentage of human CD45 expressing cells in peripheral blood of humanized NSG mice transplanted with different cell doses of either fresh or cultured HSPCs isolated from umbilical cord blood (UCB). (C) Different cell lineages in peripheral blood of mice transplanted with 1.5×105 noncultured HSPCs isolated from UCB (top) or 1.5×105 cultured HSPCs (bottom). Myeloid cells (left), B cells (middle), and T cells (right) were gated within human CD45+ cells. (D) Contribution of different cell lineages in peripheral blood at 18 weeks after transplantation. Percentages are gated within human CD45+ cells. (E) Numbers of long-term-HSC (hCD45+CD34+CD38−CD90+CD45RA−CD49f+) present in bone marrow (BM) of NSG recipients transplanted with different cell doses of either fresh or cultured HSPCs isolated from UCB. Data are represented as mean±standard deviation (SD) in (B, D, E). 50k, 0.5×105 cells; 150k, 1.5×105cells. HSPCs were isolated from a pool of seven UCB donors, three mice per group.
Mentions: For genetic modification (e.g., gene therapy) HSPCs need to be cultured for a short period. To test whether this is feasible for the NSG model, we cultured HSPCs isolated from UCB overnight to determine the effect on engraftment potential. NSG mice were transplanted with 0.5×105 or 1.5×105 HSPCs directly after isolation or after overnight culture in StemSpan supplemented with cytokines. The transplanted cells were analyzed for expression of surface markers that define human HSC subsets.14 An increase in the percentage of CD90+CD45RA− cells and, within this population, CD49f+ cells was observed after overnight culture (Fig. 1A). This increase of more primitive HSC subsets was associated with similar engraftment of human cells in lymphoid organs (Fig. 1B) and similar contribution of cell lineages to engraftment in peripheral blood between recipients of fresh or cultured HSPCs (Fig. 1C and 1D). Engraftment of human cells and lineage contribution were both unaffected by the number of transplanted cells. No differences in numbers of cells belonging to the most primitive human HSC subset were found in the BM of recipients of fresh and cultured HSPCs, although there was a trend at lower transplanted cell numbers (0.5×105, 50,000) for more primitive stem cells to be detected in the BM of recipients of cultured HSPCs (Fig. 1E). This led us to conclude that the short overnight culture, as needed for genetic modification, does not compromise engraftment potential and lineage contribution of human HSPCs in primary recipients. Furthermore, the described protocol allows for a high level of engraftment in different organs (Fig. 1B).

Bottom Line: This protocol results in robust and reproducible high levels of lympho-myeloid engraftment.Similar results were obtained with cryopreserved human bone marrow samples, thus circumventing the need for fresh cells and allowing the use of patient derived bio-bank samples.Our findings have implications for use of this model in fundamental stem cell research, immunological studies in vivo and preclinical evaluations for HSC transplantation, expansion, and genetic modification.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center , Leiden, The Netherlands .

ABSTRACT
Hematopoietic stem cells (HSCs) are defined by their ability to repopulate the bone marrow of myeloablative conditioned and/or (lethally) irradiated recipients. To study the repopulating potential of human HSCs, murine models have been developed that rely on the use of immunodeficient mice that allow engraftment of human cells. The NSG xenograft model has emerged as the current standard for this purpose allowing for engraftment and study of human T cells. Here, we describe adaptations to the original NSG xenograft model that can be readily implemented. These adaptations encompass use of adult mice instead of newborns and a short ex vivo culture. This protocol results in robust and reproducible high levels of lympho-myeloid engraftment. Immunization of recipient mice with relevant antigen resulted in specific antibody formation, showing that both T cells and B cells were functional. In addition, bone marrow cells from primary recipients exhibited repopulating ability following transplantation into secondary recipients. Similar results were obtained with cryopreserved human bone marrow samples, thus circumventing the need for fresh cells and allowing the use of patient derived bio-bank samples. Our findings have implications for use of this model in fundamental stem cell research, immunological studies in vivo and preclinical evaluations for HSC transplantation, expansion, and genetic modification.

No MeSH data available.


Related in: MedlinePlus