Our study is the first report of MTX resistance gene transfer to potentiate transplantation and selective engraftment of hESC-derived cells. We provide a model system to evaluate in vivo selection with antifolate-resistance genes in hESC-hematopoietic cells that may be applied to evaluate other chemotherapeutic drug resistance systems. In order to characterize expression of Tyr22-DHFR in hESCs, we first achieved stable DHFR-GFP expression in hESCs using three different genetic configurations. In contrast to control CSIIEG-hESCs, GFP expression decreased over time in cells transduced with DHFR-GFP bicistronic lentivirus vectors, possibly due to epigenetic silencing. We then used MTX chemotherapy and DHFR expression to establish teratomas with increased MTX resistance that were also enriched for gene-modified cells, as determined by the fold increase in transgene copies in the teratoma, compared to the GFP transgene copies detected in the undifferentiated input cell population. Finally, we demonstrated the feasibility of using Tyr22-DHFR and MTX chemotherapy to achieve in vivo selection and increased engraftment of hESC-hematopoietic cells in immunodeficient mice. These results extend our previous work on lentiviral-mediated chemoprotection in murine marrow-derived hematopoietic stem cells to demonstrate the effectiveness of this approach in ES-derived HSC and in the human system.
Historically, in vivo
selection at the level of slowly dividing stem cells has not been achieved by MTX administration alone, because the inhibitory activity of MTX affects primarily highly proliferative cells, such as myeloid and lymphoid progeny. In vivo
selection has been achieved using the anti-folate trimetrexate (TMTX) when administered along with the nucleoside transport inhibitor nitrobenzylmercaptopurine ribose phosphate (NBMPR-P) 14,45,46
. Although TMTX/NBMPR-P treatment was sufficient to achieve in vivo
selection of gene-modified HSCs in mice, selection was only transient in a study of non-human primates14,45
. In contrast to the DHFR/antifolate system, expression of P140K-O6
-methylguanine DNA methyltransferase (P140K-MGMT), which confers resistance to O6
-BG) and alkylating agents such as 1,3-bis-(2-chloroethy)-1-nitrosourea (BCNU) and temozolomide (TMZ), has been shown to support significant, long-term in vivo
selective expansion of gene-modified HSCs in dogs 7,47
. However, in a clinical study using retroviral vector to transduce MGMT activity in hematopoietic cells, the gene transfer frequency remained low 8
. The differential outcomes between the canine studies and the clinical trial highlight the importance of evaluating drug resistance genes and chemoprotection in alternative human cell sources that can be expanded ex vivo
, in order to anticipate potential challenges that may arise in future studies of large animals and humans. The differential outcomes between the antifolate studies and the MGMT studies highlight the importance of evaluating drug resistance genes and chemoprotection in alternative cell sources that can be expanded ex vivo
, in order to anticipate potential challenges that may arise in future studies of large animals and humans.
Drug resistance gene therapy models in human-mouse xenografts have been developed to determine whether high dose chemotherapy inhibits human tumor progression and the extent of protection provided by drug resistance genes (DHFR, MGMT) expressed in the mouse marrow 48,49
. Human CD34+
cells, either from umbilical cord blood (UCB) or mobilized peripheral blood (MPB) have been evaluated in mice after transfer of P140K-MGMT 50,51
. Pollok and colleagues showed that O6
-BG/BCNU treatment increased long-term engraftment of CD45+
cells in the marrow of NOD/SCID mice two to eightfold 14 weeks after transplantation of UCB or MPB CD34+
cells, respectively 50
. In Pollok’s studies, CD34-enriched cell populations that were 60% GFP+
were infused into mice that were irradiated immediately before transplantation. Four treatments of O6
-BG and BCNU had a potent selective effect in vivo
, increasing CD45+
cell engraftment in the bone marrow from 30% to 80% for UCB-derived cells and from 20% to 90% for MPB-derived cells. These results demonstrate the potential effectiveness of in vivo
human stem cell selection as xenografts in immunodeficient animals, also applicable to human ES cells as demonstrated in this article.
In our study, we transplanted an unselected population of mixed, differentiated cells that was only 20% GFP+ at the time of infusion. By normalizing GFP marking of engrafted cells to this initial percentage (i.e., by dividing the percent of GFP+ cell detected in the bone marrow by the percent of GFP+ cells in the input cell population), the selective effect of MTX treatment becomes even more apparent at twelve weeks post transplant (Figure S3). Twelve times more GFP marking (6% vs. 0.5% mean GFP+ cells) was detected in the bone marrow of HDM compared to PBS treated mice. In the peripheral blood, 5 times more GFP marking (22 % vs. 4 % mean GFP+ cells) was detected in HDM treated mice vs. PBS-treated mice. We thus achieved significantly higher long-term engraftment of hESC-hematopoietic cells in the bone marrow of treated mice, compared to untreated mice. It is also striking that the levels of CD34+/CD45+ cells were similar in LDM and HDM treatment groups. These results demonstrate the long-term selective effects of MTX on hESC-hematopoietic cell engraftment and show that MTX supports in vivo selection that persists long after treatment cessation.
iPS cells and/or hESC-derived hematopoietic cells provide candidate sources for allogeneic or autologous HSCT, so it is important to consider the conditions of this clinical translation and the potential challenges that may arise in achieving effective engraftment of these cells after transplantation. Drug resistance gene transfer represents an important complement to such cell-based therapies as a means of supporting selective engraftment of gene-modified cells for hematopoietic reconstitution and prevention of allograft rejection. In this study, we show that MTX administration provided a selective benefit to the engraftment of MTXr-DHFR hESC-hematopoietic cells in the bone marrow of NSG mice. Other conditions may improve hematopoietic differentiation 26,42,52
coupled with the evaluation of more potent drug resistance genes (such as MGMT) that also support stem cell selection will better define the potential roles of drug resistance genes in hESC-derived hematopoietic function in vivo