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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

Secondary transplantation results in high engraftment and development of all lineages. (A) Engraftment over time as measured by human CD45-expressing cells in peripheral blood of secondary recipients. (B) Development of human T cells in the thymus of secondary recipients. Gated on human CD45+ cells; ISP, immature single positive; DP, double positive; SP, single positive. (C) Development of lineages in the BM of secondary recipients. Gated on human CD45+ cells. (D) The number of cells that were transplanted into secondary recipients and (E) the number of cells that were obtained from both hind legs of secondary recipients. Gated on human CD45+ cells. Data are represented as mean±standard deviation (SD). (F) When the numbers in (D and E) are divided, this results in the fold expansion of HSCs in both hind legs of secondary recipients. Data are represented as mean±SD in (A–E). Half of total BM of three primary recipients was transplanted in two secondary recipients (donors 2 and 15) or one recipient (donor 9).
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f3: Secondary transplantation results in high engraftment and development of all lineages. (A) Engraftment over time as measured by human CD45-expressing cells in peripheral blood of secondary recipients. (B) Development of human T cells in the thymus of secondary recipients. Gated on human CD45+ cells; ISP, immature single positive; DP, double positive; SP, single positive. (C) Development of lineages in the BM of secondary recipients. Gated on human CD45+ cells. (D) The number of cells that were transplanted into secondary recipients and (E) the number of cells that were obtained from both hind legs of secondary recipients. Gated on human CD45+ cells. Data are represented as mean±standard deviation (SD). (F) When the numbers in (D and E) are divided, this results in the fold expansion of HSCs in both hind legs of secondary recipients. Data are represented as mean±SD in (A–E). Half of total BM of three primary recipients was transplanted in two secondary recipients (donors 2 and 15) or one recipient (donor 9).

Mentions: To determine the long-term repopulating ability of stem cells residing in the BM of humanized mice, we performed a secondary transplantation in a total of five mice. Half of the BM of primary recipients that had received cultured HSPCs, was thawed and transplanted via intravenous injection in one (donor 9) or two recipients (donors 2 and 15) depending on the number of cells that was recovered. Yet the recipient of donor 9 received half of the number of HSCs as compared to recipients from cells of the other two donors (Fig. 3D). Good engraftment was observed in the peripheral blood of all secondary recipients (Fig. 3A). The effect of cell number was reflected in the level of engraftment as measured in peripheral blood. Also in all secondary recipients normal development of T cells was present (Fig. 3B, for gating strategy see Supplementary Fig. S1) and all major cell lineages were present in the BM (Fig. 3C). These data show that with this protocol engraftment can be observed in secondary recipients, indicating that there was no stem cell exhaustion in the primary recipients that were transplanted with cultured HSPCs.


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)

Secondary transplantation results in high engraftment and development of all lineages. (A) Engraftment over time as measured by human CD45-expressing cells in peripheral blood of secondary recipients. (B) Development of human T cells in the thymus of secondary recipients. Gated on human CD45+ cells; ISP, immature single positive; DP, double positive; SP, single positive. (C) Development of lineages in the BM of secondary recipients. Gated on human CD45+ cells. (D) The number of cells that were transplanted into secondary recipients and (E) the number of cells that were obtained from both hind legs of secondary recipients. Gated on human CD45+ cells. Data are represented as mean±standard deviation (SD). (F) When the numbers in (D and E) are divided, this results in the fold expansion of HSCs in both hind legs of secondary recipients. Data are represented as mean±SD in (A–E). Half of total BM of three primary recipients was transplanted in two secondary recipients (donors 2 and 15) or one recipient (donor 9).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048975&req=5

f3: Secondary transplantation results in high engraftment and development of all lineages. (A) Engraftment over time as measured by human CD45-expressing cells in peripheral blood of secondary recipients. (B) Development of human T cells in the thymus of secondary recipients. Gated on human CD45+ cells; ISP, immature single positive; DP, double positive; SP, single positive. (C) Development of lineages in the BM of secondary recipients. Gated on human CD45+ cells. (D) The number of cells that were transplanted into secondary recipients and (E) the number of cells that were obtained from both hind legs of secondary recipients. Gated on human CD45+ cells. Data are represented as mean±standard deviation (SD). (F) When the numbers in (D and E) are divided, this results in the fold expansion of HSCs in both hind legs of secondary recipients. Data are represented as mean±SD in (A–E). Half of total BM of three primary recipients was transplanted in two secondary recipients (donors 2 and 15) or one recipient (donor 9).
Mentions: To determine the long-term repopulating ability of stem cells residing in the BM of humanized mice, we performed a secondary transplantation in a total of five mice. Half of the BM of primary recipients that had received cultured HSPCs, was thawed and transplanted via intravenous injection in one (donor 9) or two recipients (donors 2 and 15) depending on the number of cells that was recovered. Yet the recipient of donor 9 received half of the number of HSCs as compared to recipients from cells of the other two donors (Fig. 3D). Good engraftment was observed in the peripheral blood of all secondary recipients (Fig. 3A). The effect of cell number was reflected in the level of engraftment as measured in peripheral blood. Also in all secondary recipients normal development of T cells was present (Fig. 3B, for gating strategy see Supplementary Fig. S1) and all major cell lineages were present in the BM (Fig. 3C). These data show that with this protocol engraftment can be observed in secondary recipients, indicating that there was no stem cell exhaustion in the primary recipients that were transplanted with cultured HSPCs.

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