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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

Immunization of humanized NSG mice shows the functionality of both T cells and B cells. (A) Expansion of T cells in peripheral blood after immunization. (B) Number of CD4+ and CD8+ T cells in peripheral blood. (C) Quantification of trinitrophenyl (TNP)-specific IgG in serum of immunized mice. (D) Number of human plasma cells that were present in spleens after immunization. (E) Detection of germinal centers in spleens of immunized mice by hematoxylin and eosin staining (100× final magnification). Data are represented as mean±SD in (A–D). HSPCs were isolated from a pool of five UCB donors, three mice per group.
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f2: Immunization of humanized NSG mice shows the functionality of both T cells and B cells. (A) Expansion of T cells in peripheral blood after immunization. (B) Number of CD4+ and CD8+ T cells in peripheral blood. (C) Quantification of trinitrophenyl (TNP)-specific IgG in serum of immunized mice. (D) Number of human plasma cells that were present in spleens after immunization. (E) Detection of germinal centers in spleens of immunized mice by hematoxylin and eosin staining (100× final magnification). Data are represented as mean±SD in (A–D). HSPCs were isolated from a pool of five UCB donors, three mice per group.

Mentions: To test the functionality of both T and B cells, we immunized the mice that were transplanted with cultured human HSPCs with TNP-KLH, a T cell–dependent B-cell antigen. One week after the last challenge an increase in the number of T cells in the peripheral blood of immunized mice was observed as compared to nonimmunized mice (Fig. 2A). This increase in number of T cells was caused by an expansion of CD4+ T helper cells, which are the cells to provide crosstalk to B cells (p<0.05, Fig. 2B). To determine the functionality of the B cells, an ELISA for TNP-specific antibodies was performed. An increase of TNP-specific IgG was only measured in the serum of immunized animals (Fig. 2C). In addition, more plasma cells were observed in the spleens, but not in the BM, of immunized animals as compared to nonimmunized mice (Fig. 2D). Germinal centers could also be detected by hematoxylin and eosin staining of sections of the spleen of immunized mice (Fig. 2E). In conclusion, immunization of young NSG mice transplanted with overnight cultured human HSCs showed that both T cells and B cells are functional.


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)

Immunization of humanized NSG mice shows the functionality of both T cells and B cells. (A) Expansion of T cells in peripheral blood after immunization. (B) Number of CD4+ and CD8+ T cells in peripheral blood. (C) Quantification of trinitrophenyl (TNP)-specific IgG in serum of immunized mice. (D) Number of human plasma cells that were present in spleens after immunization. (E) Detection of germinal centers in spleens of immunized mice by hematoxylin and eosin staining (100× final magnification). Data are represented as mean±SD in (A–D). HSPCs were isolated from a pool of five UCB donors, three mice per group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4048975&req=5

f2: Immunization of humanized NSG mice shows the functionality of both T cells and B cells. (A) Expansion of T cells in peripheral blood after immunization. (B) Number of CD4+ and CD8+ T cells in peripheral blood. (C) Quantification of trinitrophenyl (TNP)-specific IgG in serum of immunized mice. (D) Number of human plasma cells that were present in spleens after immunization. (E) Detection of germinal centers in spleens of immunized mice by hematoxylin and eosin staining (100× final magnification). Data are represented as mean±SD in (A–D). HSPCs were isolated from a pool of five UCB donors, three mice per group.
Mentions: To test the functionality of both T and B cells, we immunized the mice that were transplanted with cultured human HSPCs with TNP-KLH, a T cell–dependent B-cell antigen. One week after the last challenge an increase in the number of T cells in the peripheral blood of immunized mice was observed as compared to nonimmunized mice (Fig. 2A). This increase in number of T cells was caused by an expansion of CD4+ T helper cells, which are the cells to provide crosstalk to B cells (p<0.05, Fig. 2B). To determine the functionality of the B cells, an ELISA for TNP-specific antibodies was performed. An increase of TNP-specific IgG was only measured in the serum of immunized animals (Fig. 2C). In addition, more plasma cells were observed in the spleens, but not in the BM, of immunized animals as compared to nonimmunized mice (Fig. 2D). Germinal centers could also be detected by hematoxylin and eosin staining of sections of the spleen of immunized mice (Fig. 2E). In conclusion, immunization of young NSG mice transplanted with overnight cultured human HSCs showed that both T cells and B cells are functional.

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