Expanded CD34+ human umbilical cord blood cells generate multiple lymphohematopoietic lineages in NOD-scid IL2rgamma(null) mice

Abstract

Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation, we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells, cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid, B-lymphoid, and erythroid lineages, but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization, which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice.

Figure 1

Lineage marker expression on engrafted human CD45⁺ cells in NOD-scid IL2rγ^null mice. NOD-scid IL2rγ^null mice were transplanted with 1 × 10⁶ cultured CD34⁺ human UCB cells. Bone marrow was analyzed by flow cytometry for the presence of human myeloid and lymphoid lineages three (A) and twelve (B) weeks after transplantation and spleen was analyzed twelve weeks (C) after transplantation. Total human cells are shown as the percent positive human CD45⁺ cells. Lineage markers are expressed as percent of human CD45⁺ cells in the bone marrow. CD71 and GlyA populations are expressed as percent of murine CD45⁻ cells. Shown are representative histograms.

Figure 2

Engraftment of human CD45⁺ cells in non-TNFα-treated and TNFα-treated NOD-scid IL2rγ^null mice 12 weeks after transplantation of cultured human UCB. Irradiated non-TNFα-treated and TNFα-treated NOD-scid IL2rγ^null mice were transplanted with 1 × 10⁶ cultured CD34⁺ human UCB cells. (A) Percent and (B) number of human CD45⁺ cells in blood, bone marrow, spleen, and thymus twelve weeks after transplantation. *P < 0.025, **P < 0.01, ***P < 0.001.

Figure 3

Phenotype of human CD45⁺ cells in the thymus of non-TNFα-treated and TNFα-treated NOD-scid IL2rγ^null twelve weeks after transplantation of cultured human UCB. Irradiated non-TNFα-treated and TNFα-treated NOD-scid IL2rγ^null mice were transplanted with 1 × 10⁶ cultured CD34⁺ human UCB cells. Twelve weeks later, thymi were analyzed by flow cytometry. Representative flow cytometric profiles of human CD45⁺ cell development in (A) non-TNFα-treated and (B) TNFα-treated NOD-scid IL2rγ^null mice. Human CD3 percentages are expressed as percent human CD45⁺ cells. Human CD4 and CD8 percentages are expressed as percent human CD3⁺ cells.

Figure 4

Immunohistochemistry of thymi of TNFα-treated NOD-scid IL2rγ^null mice twelve weeks after transplantation of cultured human UCB. Irradiated TNFα-treated NOD-scid IL2rγ^null mice were transplanted with 1 × 10⁶ cultured CD34⁺ human UCB cells. Twelve weeks later, thymi were analyzed by immunohistochemistry. Thymic sections of TNFα-treated mice contained human CD45⁺ cells (A), which were localized predominantly in the subcapsular region. Many of these CD45⁺ cells were CD5⁺ (B) and expressed either CD4⁺(C) and/or CD8⁺ (D). In addition, CD1a⁺ thymocytes were detected residing in the cortex of the thymus (E). TdT⁺ (F) and CD99⁺ (G) thymocytes were also present, as were numerous human CD45⁺ cells that expressed the proliferation antigen Ki-67 (H).

Figure 5

Human CD45⁺ cell development in the spleen of non-TNFα-treated and TNFα-treated NOD-scid IL2rγ^null mice twelve weeks after transplantation of cultured human UCB. Irradiated non-TNFα-treated and TNFα-treated NOD-scid IL2rγ^null mice were transplanted with 1 × 10⁶ cultured CD34⁺ human UCB cells. Twelve weeks later, spleen cells were analyzed by flow cytometry. (A) Percentage of human CD3⁺ and CD20⁺ cells in the spleen. Representative flow cytometry profiles of human cell development in (B) non-TNFα-treated and (C) TNFα-treated NOD-scid IL2rγ^null mice. Lineage markers expressed as percent of human CD45⁺ cells. Human CD4 and CD8 percentages are expressed as percent of human CD3⁺ cells.

Figure 6

Immunohistochemistry of spleens of TNFα-treated NOD-scid IL2rγ^null mice twelve weeks after transplantation of cultured human UCB. TNFα-treated NOD-scid IL2rγ^null mice were transplanted with 1 × 10⁶ cultured CD34⁺ human UCB cells. Twelve weeks later, the distribution of human lymphocytes in the spleen was determined by immunohistochemistry. Immunoperoxidase staining of human (A) CD45⁺, (B) CD20⁺, (C) CD5⁺; (D, arrows) CD138⁺, (E, arrows) kappa⁺ and (F, arrows) lambda⁺ cells in spleens of TNFα-treated NOD-scid IL2rγ^null mice.

Figure 7

Human immunoglobulin levels and T-independent immune responses in TNFα-treated NOD-scid IL2rγ^null mice twelve weeks after transplantation of cultured human UCB. TNFα-treated NOD-scid iL2rγ^null mice were transplanted with 1 × 10⁶ cultured CD34⁺ human UCB cells. Represented by the dashed line in each panel is the upper limit of the mean of pre-immune sera plus 3 standard deviations. All values above this limit were considered “responders.” The treatment group and the number of responders for each treatment group are shown at the bottom of each panel. Each symbol represents an individual animal. Panel A: Human IgG and IgM antibodies prior to and following Pneumovax immunization in the presence or absence of BLyS treatment. Panel B: Human IgG and IgM antibodies to serotype 4 and 14 following Pneumovax immunization in the presence or absence of BLyS treatment. Each symbol represents and individual animal. *P<0.05, **P<0.001.

Figure 8

Human CD3 proliferation and human T-dependent immune responses in TNFα-treated NOD-scid IL2rγ^null mice twelve weeks after transplantation of cultured human UCB. TNFα-treated NOD-scid IL2rγ^null mice were transplanted with 1 × 10⁶ cultured CD34⁺ human UCB cells. Panel A: Proliferation of human CD3 T cells following ex vivo stimulation with anti-CD3 twelve weeks after transplantation. Shown are results of triplicate cultures of spleen cells from individual mice displayed as mean ± SD. Panel B: Human IgG antibodies to tetanus following immunization with tetanus toxoid. Each symbol represents an individual animal. Represented by the dashed line in each panel is the upper limit of the mean of pre-immune sera plus 3 standard deviations. All values above this limit were considered “responders.” The treatment group and the number of responders for each treatment group are shown at the bottom of the panel. Each symbol represents an individual animal.