Bone marrow-derived mesenchymal stem cells (MSCs) are known to interact with hematopoietic stem cells (HSCs) and immune cells, and represent potential cellular therapy to enhance allogeneic hematopoietic engraftment and prevent graft-versus-host disease (GVHD). We investigated the role of human MSCs in NOD-SCID mice repopulation by unrelated human hematopoietic cells and studied the immune interactions between human MSCs and unrelated donor blood cells in vitro. When hematopoietic stem cell numbers were limited, human engraftment of NOD-SCID mice was observed only after coinfusion of unrelated human MSCs, but not with coinfusion of mouse mesenchymal cell line. Unrelated human MSCs did not elicit T-cell activation in vitro and suppressed T-cell activation by Tuberculin and unrelated allogeneic lymphocytes in a dose-dependent manner. Cell-free MSC culture supernatant, mouse stromal cells and human dermal fibroblasts did not elicit this effect. These preclinical data suggest that unrelated, human bone marrow-derived, culture-expanded MSCs may improve the outcome of allogeneic transplantation by promoting hematopoietic engraftment and limiting GVHD and their therapeutic potential should be tested in clinic.

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ABSTRACT

We report the first clinical trial that used a cross-over design showing that high-dose cyclophosphamide plus G-CSF results in mobilization of more progenitors then GM-CSF plus G-CSF when tested in the same patient regardless of sequence of administration, although the regimen is associated with greater morbidity. Patients who fail to achieve adequate mobilization after regimen G can be treated with regimen C as an effective salvage regimen, whereas patients who fail regimen C are unlikely to benefit from subsequent treatment with regimen G. The cross-over design allowed detection of significant differences between regimens in a small cohort of patients and should be considered in design of future comparisons of mobilization regimens.

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Human hematopoietic progenitors express low levels of O 6 -alkylguanine-DNA alkyltransferase and are sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), particularly following O 6 -benzylguanine (BG)-mediated O 6 -alkylguanine-DNA alkyltransferase inhibition. Expression of the BG-resistant mutant (G156A) methylguanine methyltransferase (⌬MGMT ) gene in hematopoietic cells confers resistance to BG and BCNU. Because BCNU targets both early and late human hematopoietic cells and results in prolonged and cumulative myelosuppression, we attempted to protect early hematopoietic progenitors (long-term culture initiating cells (LTC-ICs)) by retroviral-mediated transfer of the ⌬MGMT gene. A total of 33-56% of LTC-ICs were transduced with MFG-⌬MGMT retrovirus as determined by evidence of provirus in secondary colony-forming units at 5 weeks of culture under conditions optimal for the survival and proliferation of early hematopoietic progenitors. The addition of flt-3 ligand to cultures increased the transduction rate of LTC-ICs. Furthermore, 17.8 Ϯ 8.1% of ⌬MGMT-transduced LTC-ICs survived doses of BG and BCNU; these doses allowed the survival of only 0 -1% of untransduced LTC-ICs. This finding compares favorably with the 8 -12% of CD34 ϩ cell-derived colony-forming units that we previously showed became resistant to BG and BCNU after ⌬MGMT gene transfer. Thus, ⌬MGMT transduction of human early hematopoietic progenitor LTC-ICs confers resistance to BG and BCNU and may allow transduced LTC-ICs selective survival and enrichment over untransduced cells in patients undergoing BG and BCNU chemotherapy.

Cytokine-based expansion of umbilical cord blood (UCB) in vitro prior to infusion has been pursued in an attempt to overcome the limited cellular content of a single UCB unit. Thus far, these attempts have not shown improvement in kinetics of donor-derived hematopoietic recovery. Our studies have incorporated UCB expanded over a feeder-layer of human mesenchymal stem cells (huMSC), known to inhibit the differentiation of hematopoietic stem cells (HSC) observed in expansion with cytokines alone. Expansion conditions included: UCB expanded over a huMSC monolayer with the addition of cytokines (IL-3, IL-6, G-CSF, SCF, FLT-3L, EPO) and UCB expanded in the same cytokines alone. Day 12 culture readouts included: viable cell counts, 4-color flow analysis, and rates of human engraftment in NOD/SCID mice. In the current study the fold expansion was 6.4 fold in the huMSC + cytokines condition and 7 fold in the cytokines alone condition. Flow cytometry surface marker analysis proportions (absolute numbers) were notable for higher proportions and numbers of early HSC expressing CD133 in cultures incorporating huMSC stromal layer: Unexpanded MSC+ cytokines Cytokines CD34 0.68 (.068M) 0.74 (3.63M) 1.94 (5.39M) CD133 5.69 (.569M) 2.56 (12.54M) 0.74 (2.06M) CD3 49.6 (4.96M) 2.2 (10.78M) 0.42 (1.17M) CD56 17.4 (1.74M) 2.71 (13.28M) 1.06 (2.95M) CD69 0.80 (7.28M) 7.28 (35.67M) 24.4 (67.8M) UCB graft T and NK populations were maintained in huMSC culture conditions and the observed difference in CD69 expression supports the hypothesis that huMSC may have an inhibitory effect on T cell activation during UCB ex vivo expansion. To assess the human engraftment potential of the cultures, cells from each culture condition were injected by tail vein into NOD/SCID mice (no CD34 selection was performed). Mice receiving unexpanded UCB received 10M mononuclear cells each. Mice receiving culture expanded cells received cell doses in proportion to the fold expansion over the number of cells at the initiation of the cultures. Engraftment was assessed by the percentage of human CD45+ (≥0.4%) cells found within the bone marrow of mice at seven weeks post infusion. Mice were injected as follows: 7 mice with unexpanded UCB (2 of which died within a month of transplant), 7 mice with UCB expanded in huMSC + cytokines, and 3 mice with UCB expanded in cytokines alone. Flow analysis of mouse bone marrow cells revealed average CD45+ percentages of 1.79% for mice injected with unexpanded UCB, 2.66% for mice injected with cytokine alone cells, and 5.94% for mice injected with huMSC + cytokine cells. Human cell subset analysis was performed for CD3, CD19, and CD56 content. The percentages of gated CD45+ co-expressing CD3+ were 10.3% in the unexpanded UCB, 16.6% in the cytokine alone condition and 10.4% in the huMSC + cytokine condition. Cells co-expressing CD19+ were 7.86% in the unexpanded UCB, 8.31% in the huMSC + cytokine condition and dropped to 1.43% in the cytokine alone condition. Gated CD45+ cells co-expressing CD56+ were 16.4% in the unexpanded UCB, 8.8% in the huMSC + cytokines condition, and dropped to 2.6% in the cytokines alone condition. In conclusion, UCB expanded short-term in cytokines demonstrates maintenance of earlier HSC phenotype and improved human engraftment in NOD/SCID in cultures incorporating a huMSC monolayer platform.

Background. Umbilical cord blood (UCB), a source of hematopoeitic stem cells (HSC), is marked by delayed engraftment attributed to the limited cellular content of a single UCB unit. Cytokine-based ex vivo expansion of UCB is a way of increasing the number of cells available for allogeneic transplants, however, this strategy has not demonstrated improved engraftment in human clinical trials. Further studies have incorporated human mesenchymal stem cells (huMSC) which may provide signals that control the proliferation, survival, and differentiation of HSC. In attempt to reduce the occurrence and severity of GVHD following allogeneic transplants, strategies such as utilizing T-cell depleted grafts have been pursued, however, clinical trials using these grafts have shown decreased rates of donor engraftment, suggesting the requirement of accessory cells as well as HSC to achieve engraftment. Methods. UCB mononuclear cells (MNC) were cultured using cytokines (IL-3, IL-6, G-CSF, SCF, Flt-3L, EPO) with or without a feeder-layer of huMSC for 12 days. On day 12, viability, 4-color flow cytometry, and human engraftment potential were measured. Human engraftment potential was determined by injecting cells (without CD34+ selection) from each culture condition and non-cultured UCB MNC, via tail vein, into sublethal irradiated NOD/SCID mice. Mice were injected with unexpanded UCB MNC (n=23), UCB expanded in huMSC+cytokines (n=21) and UCB expanded in cytokines alone (n=10). 7–9 weeks following injection of human cells, bone marrow was harvested and analyzed for human content. Positive human engraftment was determined by a human %CD45+ of ≥ 0.4%. Results. An 8.77 fold expansion of UCB cultured in cytokines alone compared to a 7.14 fold expansion of UCB cultured in huMSC + cytokines was observed. Surface phenotyping of expanded UCB, and human cells emerging in the bone marrow of NOD/SCID mice following injection of cultured and non-cultured UCB are in Table 1. Unexpanded huMSC+cytokines Cytokines % CD3 44.0 (4.40M) 2.04 (10.1M) 1.52 (4.35M) % CD56 17.0 (1.70M) 7.07 (36.1M) 3.69 (13.8M) % CD34 3.41 (.341M) 2.25 (10.9M) 3.52 (12.3M) Bone marrow of NOD/SCID mice % CD45+ 2.57 3.58 2.38 % of CD45+ co-expressing CD3 11.0 9.25 18.7 % of CD45+ co-expressing CD19 33.5 16.6 19.4 % of CD45+ co-expressing CD56 10.1 8.04 1.60 Human engraftment was seen in 13 mice which received unexpanded UCB, 10 mice which received UCB expanded in huMSC+cytokines and only 3 mice which received UCB expanded in cytokines alone. Statistical analysis, using multivariable logistic regression to determine the factors that predict engraftment, revealed that the proportions of T and NK cells present in expanded UCB correlated with engraftment. A 10% increase in the proportion of CD45+ co-expressing CD3 was associated with a 1.79 fold increase in engraftment (p=0.016), whereas each 10% increase in the proportion of CD45+ co-expressing CD56 increased the odds of engrafting by 104% (p= 0.003). Conclusions. We observed an expansion of CD34 hematopoietic progenitors as well as a greater proportion of CD3+ cells, in expansion conditions incorporating huMSC. Additionally, we observed improved rates of engraftment in this expansion condition. Therefore, although the mechanism by which accessory cells including T and NK cells facilitate HSC engraftment is not known, we observed that the presence of accessory cells in addition to CD34 hematopoietic progenitors facilitated engraftment in NOD/SCID mice.