Limits...
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

Transplantation of overnight cultured HSCs from both cryopreserved UCB and cryopreserved BM resulted in the development of all lymphoid lineages and high human chimerism. (A) Engraftment in peripheral blood of human CD45+ (hCD45+) cells in mice transplanted with different cell doses of HSCs isolated from UCB or human BM. Contribution of different lineages in peripheral blood of NSG mice transplanted with 150,000 HSCs obtained from UCB (B) or 200,000 HSCs obtained from BM (C). Gated on human CD45+ cells. UCB: three different pools of at least two donors in a total of 13 recipients; BM: three different donors in a total of 13 recipients.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4048975&req=5

f4: Transplantation of overnight cultured HSCs from both cryopreserved UCB and cryopreserved BM resulted in the development of all lymphoid lineages and high human chimerism. (A) Engraftment in peripheral blood of human CD45+ (hCD45+) cells in mice transplanted with different cell doses of HSCs isolated from UCB or human BM. Contribution of different lineages in peripheral blood of NSG mice transplanted with 150,000 HSCs obtained from UCB (B) or 200,000 HSCs obtained from BM (C). Gated on human CD45+ cells. UCB: three different pools of at least two donors in a total of 13 recipients; BM: three different donors in a total of 13 recipients.

Mentions: We tested whether our optimized protocol gave good engraftment of HSPCs from cryopreserved BM and development of different lymphoid lineages. Different cell doses of overnight cultured HSPCs from UCB and BM were transplanted in NSG mice. For both HSPC sources it was observed that there was a higher engraftment when more cells were transplanted (Fig. 4A). Although engraftment was consistently higher in mice transplanted with cryopreserved UCB, we observed good repopulation in mice transplanted with cells obtained from cryopreserved BM but higher cell doses were needed for robust engraftment compared to UCB. There was no difference observed in lineage contribution in peripheral blood between the two cell sources (Fig. 4B and 4C). These data show that with our optimized protocol it is possible to get good engraftment and development of all lineages in NSG mice transplanted with HSPCs isolated from cryopreserved human BM samples.


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)

Transplantation of overnight cultured HSCs from both cryopreserved UCB and cryopreserved BM resulted in the development of all lymphoid lineages and high human chimerism. (A) Engraftment in peripheral blood of human CD45+ (hCD45+) cells in mice transplanted with different cell doses of HSCs isolated from UCB or human BM. Contribution of different lineages in peripheral blood of NSG mice transplanted with 150,000 HSCs obtained from UCB (B) or 200,000 HSCs obtained from BM (C). Gated on human CD45+ cells. UCB: three different pools of at least two donors in a total of 13 recipients; BM: three different donors in a total of 13 recipients.
© Copyright Policy
Related In: Results  -  Collection

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

f4: Transplantation of overnight cultured HSCs from both cryopreserved UCB and cryopreserved BM resulted in the development of all lymphoid lineages and high human chimerism. (A) Engraftment in peripheral blood of human CD45+ (hCD45+) cells in mice transplanted with different cell doses of HSCs isolated from UCB or human BM. Contribution of different lineages in peripheral blood of NSG mice transplanted with 150,000 HSCs obtained from UCB (B) or 200,000 HSCs obtained from BM (C). Gated on human CD45+ cells. UCB: three different pools of at least two donors in a total of 13 recipients; BM: three different donors in a total of 13 recipients.
Mentions: We tested whether our optimized protocol gave good engraftment of HSPCs from cryopreserved BM and development of different lymphoid lineages. Different cell doses of overnight cultured HSPCs from UCB and BM were transplanted in NSG mice. For both HSPC sources it was observed that there was a higher engraftment when more cells were transplanted (Fig. 4A). Although engraftment was consistently higher in mice transplanted with cryopreserved UCB, we observed good repopulation in mice transplanted with cells obtained from cryopreserved BM but higher cell doses were needed for robust engraftment compared to UCB. There was no difference observed in lineage contribution in peripheral blood between the two cell sources (Fig. 4B and 4C). These data show that with our optimized protocol it is possible to get good engraftment and development of all lineages in NSG mice transplanted with HSPCs isolated from cryopreserved human BM samples.

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