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Abstract

Gene therapy as well as methods capable of returning cells to a pluripotent state (iPS) have enabled the correction of genetic deficiencies in syngenic adult progenitors, reducing the need for immunosuppression in cell therapy approaches. However, in diseases involving mutations that lead to the complete lack of a protein, such as Duchenne muscular dystrophy, the main immunogens leading to rejection of transplanted cells are the therapeutic proteins themselves. In these cases even iPS cells would not circumvent the need for immunosuppression, and alternative strategies must be developed. One such potential strategy seeks to induce immune tolerance using hematopoietic stem cells originated from the same donor or iPS line from which the therapeutic progenitors are derived. However, donor hematopoietic stem cells (HSCs) are available in limiting numbers and embryonic stem (ES) cell-derived HSCs engraft poorly in adults. While these limitations have been circumvented by ectopic expression of HOXB4, overexpression of this protein is associated with inefficient lymphoid reconstitution. Here we show that adult HSCs expanded with a NUP98-HOXA10hd fusion protein sustain long-term engraftment in immunologically mismatched recipients and generate normal numbers of lymphoid cells. In addition, NUP98-HOXA10hd-expanded cells induce functional immune tolerance to a subsequent transplant of myogenic progenitors immunologically matched with the transplanted HSCs.

Keywords

HOXA10 Chimerism Hematopoietic stem cell (HSC) and myoblast transplantation Tolerance

Introduction

Duchenne muscular dystrophy (DMD) and related forms of muscular diseases are characterized by severe muscle damage, often leading to premature death. Current treatments that aim to extend a patient's mobility and survival are insufficient. Among the potential new therapies for DMD, one of the most promising clinical approaches relies on intramuscular transplantation of muscle stem cells from a healthy donor (10,14,16,22). Satellite cell allotransplantation, like any other allograft, raises the problem of immune recognition and rejection of the therapeutic donor cells, necessitating immunosuppression in order to facilitate efficient muscle engraftment (5,8). Induced pluripotent stem (iPS) cells could offer novel alternatives to circumvent allogenic transplant involving immunosuppression treatment. However, in the case of DMD, where dystrophin is absent, the transplantation of wild-type cells has been shown to trigger an immune reaction due to the immunogenicity of the wild-type protein itself. Therefore, independent of the source of donor cells, for DMD and a number of similar genetic conditions in which the therapeutic gene product is seen as non-self by the patient's immune system, long-term blockade of the immune response is required. To this end, the induction of immune tolerance represents a viable strategy.

Currently, the main approach used to dampen immune rejection uses immunosuppressive drugs, which have significant side effects, including tumor growth, infections, metabolic disorders, or organ damage. An alternative to this pharmacological intervention is induction of donor-specific tolerance by bone marrow (BM) transplantation (24). Stable hematopoietic chimerism can lead to thymic and peripheral deletion of lymphocytes reactive towards either donor or host, which results in long-term tolerance and limited graft-versus-host disease (GVHD). Such a protocol has been in existence for more than 50 years (4) and has been utilized successfully in several solid organ transplants (7). Despite the recognition that hematopoietic stem cell (HSC) transplants can lead to robust tolerance, myeloablative conditioning for HSC engraftment, generally used for diseases such as leukemia, is considered too toxic for routine use in organ transplantation situations. The only protocols currently accepted in such cases are noncytoreductive and usually require larger numbers of HSCs than are routinely available in order to obtain chimerism (24). Two alternative approaches may circumvent the problems caused by the limiting numbers of HSCs. The first entails in vitro expansion of HSCs, although, after years of focused research, our ability to expand unmodified HSCs in vitro is still severely limited. The second approach is based on the use of embryonic stem (ES) cells, which can be expanded to large numbers and induced to form tissue-committed adult-type progenitors, including HSCs. The limitation to this method lies in the inability of ES-derived HSCs to efficiently reconstitute BM of adult recipients (12). We have addressed the challenges imposed by either approach by engineering HSCs that overexpress transcription factors of the HOX family or their derivatives.

The HOX family of homeobox genes has been shown to play an important role in hematopoiesis (2). Several studies have reported that overexpression of HOXB4 induces the rapid expansion of HSCs ex vivo. Furthermore, HOXB4 confers the ability upon donor cells to fully replenish the numbers of HSCs normally present in BM without expanding beyond the normal limits, suggesting that HOXB4-expressing cells are still subject to physiological mechanisms controlling their proliferation (1,17,26). Finally, recent studies have shown that the expression of HOXB4 in ES-derived hematopoietic cells enables their engraftment in adult recipients (11). The primary drawback of using HOXB4 to overcome limitations in HSC availability or engraftment properties stems from the fact that high levels of this transcription factor skew hematopoietic progenitor differentiation towards myeloid lineages, resulting in poor reconstitution of the lymphoid compartment in transplanted recipients (18). Therefore, we sought to examine the ability of other members of the HOX protein family to reconstitute the hematopoietic compartment with normal myeloid/lymphoid lineage ratios. Recent work on NUPA10hd, a fusion of the N-terminal domain of nucleoporin 98 (NUP98) and the homeodomain only of another member of the HOX transcription factor family (HOXA10), has shown promising results in BM reconstitution. Sekulovic et al. were able to induce more than 1000-fold expansion of HSCs in the short-term in vitro culture and, in contrast to HOXB4, the HOXA10hd fusion protein NUPA10hd-infected HSCs demonstrate a high capacity for BM repopulation without inducing an imbalance between myeloid and lymphoid cell production (13,19).

Here we explore the ability of NUPA10hd-expanded HSCs to induce functional immune tolerance. Our data show that expanded NUPA10hd-infected HSCs can efficiently reconstitute fully immunologically mismatched recipient mice and that the resulting chimeric mice display long-term tolerance to the transplantation of myogenic progenitor cells immunologically matched with the transplanted HSCs.

Materials and Methods

Retroviral Vectors

The generation of MSCV-NUP98-HOXA10hd-IRES-GFP (NUPA10hd) viral vector has been described previously (15).

Animals

CD45.1, CD45.2, and GFP+CD45.2 C57BL/6 mice (H-2^b) were bred in-house and maintained in a pathogen-free environment. GFP⁺CD45.2 C57BL/6 mice express GFP ubiquitously from the cytomegalovirus enhancer-chicken β-actin hybrid promoter (28). Balb/c mice were purchased from Jackson Laboratories. C57BL/6 CD45.2 mice, 4–6 weeks of age, were used as donors for bone marrow transplantation. GFP+CD45.2 C57BL/6 mice, 8–10 weeks of age, were used as donors for satellite cell transplantation. C57BL/6 CD45.1 mice, 12 weeks of age, were used as syngenic hosts for control groups and age-matched Balb/c mice were used as allogenic hosts. All experiments were approved by the University of British Columbia Animal care committee.

Antibodies and Staining Reagent for FACS Sorts

Anti-Gr-1, anti-α7 integrin, and anti-CD3 monoclonal antibodies were purified using hybridoma maintained in our laboratory and respectively conjugated to PE (Prozyme) and to Alexa 647 (Invitrogen) according to the manufacturer's instructions. Antibodies against H-2K^b conjugated to biotin, anti-C-kit conjugated to PE, and anti-CD45 conjugated to PerCP were purchased from BD Biosciences Pharmingen; anti-Mac-1 conjugated to PE, anti-CD3, B220, Mac-1, TER119, Gr-1 conjugated to PE-Cy7, anti-CD45.2, Sca-1 conjugated to either APC or PE-Cy7 were purchased from eBioscience. Streptavidin conjugated to APC was purchased from Caltag. Hoechst was purchased from Sigma.

Purification of Lin^–Sca⁺C-kit⁺ (LSK) Cells

Lin⁺ cells were depleted by staining bone marrow (BM) with antibodies to CD3, B220, Mac-1, TER119, Gr-1 (all conjugated to PE-Cy7) and anti-PE magnetic beads followed by separation on an autoMACS (Miltenyi Biotec). The Lin^–-enriched fraction was stained with anti-C-kit-PE and Sca-1-APC. Sorting was performed on a FACSVantage (Becton Dickinson).

Infection of LSK Cells with NUPA10hd Retrovirus

Cultures were initiated with sorted LSK mouse bone marrow cells. After isolation, cells were prestimulated in the presence of serum and hematopoietic growth factors for a day, exposed to NUP98-HOXA10hd-IRES-GFP (NUPA10hd) retrovirus for 48 h, and then maintained in vitro for 6 more days as previously described (13,19).

Transplant of Infected Cells and Flow Cytometry Analysis

Infected LSK cells were collected 8 days after infection and sorted for GFP⁺ on a FACSVantage (Becton Dickinson). Recipient mice were subjected to a sublethal 8.5 Gy cobalt-60 gamma irradiation dose and received 30–300,000 cells intravenously. The group injected with 30 cells also received 300,000 Sca^– cells generated by magnetic depletion of BM from C57BL/6-CD45.1 mice. For chimerism analyses, peripheral blood was collected from the saphenous vein into heparinized tubes and red blood cells were lysed with an ammonium chloride solution. The remaining white blood cells were stained with anti-H-2K^b-biotin, followed by streptavidin-APC, and Gr-1-PE, CD3-PE-Cy7 or Mac-1-PE, B220-PE-Cy7. For control groups (C57BL/6-CD45.1 hosts) anti-H-2K^b-biotin was replaced by CD45.2-APC.

Satellite Cell Sort and Transplantation

Muscle cells from GFP⁺ C57BL/6 mice were isolated and myoblasts were sorted as described previously (9). Briefly, isolated muscle cells were stained with anti-Sca-1-PE Cy7, anti-α7 integrin-Alexa 647, anti-CD31-and anti-CD45-PerCP, and sorted on a FACSVantage (Becton Dickinson); 30,000 α7 integrin-positive, CD31-, CD45-, and Sca-1-negative cells were injected directly into the tibialis anterior in 20 μl PBS. Transplanted muscles were harvested and analyzed by immunohistochemistry 4 weeks after transplant.

Tissue Analysis

Mice were terminally anesthetized and spleens were harvested for mixed lymphocyte reaction (MLR) assays. Then, mice were perfused with 50 ml of PBS and with 20 ml of 4% paraformaldehyde in PBS. Limb muscles were collected and postfixed for 2 h in 2% paraformaldehyde and stored overnight in 20% sucrose at 4°C for cryoprotection. The tissues were embedded in Tissue-Tek OCT, frozen on dry ice, and stored at −80°C until processing. Samples were stained with Hoechst and visualized using a Zeiss Axioplan 2 microscope. Images were digitally captured using a charge-coupled device (QImaging) and analyzed using Openlab software (Improvision).

Mixed Lymphocyte Reactions (MLR)

Spleens were aseptically removed from terminally anesthetized mice before perfusion or fixatives were applied. Tissue was stored in complete media on ice until processing (DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% L-glutamine, 1% nonessential amino acid solution, 1% sodium pyruvate, and 0.1% β-mercaptoethanol). Individual spleens were dissociated by pushing through a 70-μM nylon filter (BD Pharmingen) with a syringe plunger. The filter was washed and the recovered cells pelleted in a centrifuge at 1500 rpm for 5 min at 4°C. The pellet was resuspended in Tris-NH₄Cl-based cell lysis buffer, incubated for 10 min at room temperature, when the reaction was stopped with 10% serum and the cells washed and counted. Stimulator cells (antigen presenting cells, or APC) of C57BL/6 origin were irradiated with 1500 rads in a Gammacell irradiator. Cells were then plated in each well of flat bottom 96-well tissue culture plates (Nunc). To assess tolerance under coculture conditions, 2 × 10⁵ responder cells were cultured in the presence of 10⁴, 5 × 10⁴, and 10⁵ stimulator cells. Controls included wells containing no stimulator cells, stimulator cells alone, and anti-CD3 (2C11, in house) mitogen stimulation. The cultures were incubated at 37°C, 5% CO₂ for 72 h, when they were pulsed with 1 μCi of [³H]TdR and returned to the incubator for a final 24 h. Cultures were harvested on to glass filters and subsequently read on a 96-well format Top Count machine. All samples were plated in triplicate and the graphs reflect average cpm ([³H]TdR incorporation) ± SE.

Results

Transplantation of BM Stem/Progenitor Cells Expressing NUPA10hd Induces Stable Hematopoietic Chimerism in Allogeneic Recipients

NUPA10hd-expressing HSCs can undergo multilog expansion in short-term liquid culture and subsequently efficiently reconstitute isogenic recipients. However, the performance of NUPA10hd-expanded cells in allogeneic transplants has not been explored. Therefore, we set out to determine whether C57BL/6 (H-2^b) NUPA10hd-expressing HSCs would effectively generate stable hematopoietic chimerism in fully MHC-mismatched Balb/c (H-2^d) recipients. The diagram in Figure 1A shows the experimental design. FACS-purified LSK stem/progenitor cells were infected with a retrovirus expressing NUPA10hd as well as GFP. Cultures were initiated with 1.6 × 10⁵ purified LSK cells (~55% GFP⁺) (Fig. 1B) or ~3,000 transduced HSCs (based on HSC frequency of 1/30 for LSK). After 8 days, the total cell number had increased by over 300-fold with commensurate expansion of transduced HSC content (13). Viable GFP⁺ cells were purified by flow cytometry and transplanted into sublethally irradiated Balb/c mice (8.5 Gy). To assess the minimum number of NUPA10hd-expanded cells required to efficiently achieve chimerism in allogeneic Balb/c recipients, five doses of sorted cells ranging from 1/1,000,000th of the culture to 1/100th of the culture were transplanted. Based on an estimated HSC expansion of 300-fold, this represents a transplant dose range of 0.9 HSCs to 9,000 HSCs, respectively (corresponding to starting equivalent of 0.003 HSCs to 30 HSCs). A parallel control group of Balb/c recipients received 3,000 freshly sorted, uninfected C57BL/6 LSK cells (corresponding to an estimated 100 HSCs).

Figure 1.

LSK cells are efficiently transduced by NUPA10hd. (A) Experimental design of BM transplantation. (B) Dot plot shows the proportion of GFP-positive LSK cells, transduced with NUPA10hd-GFP.

Twelve weeks after transplant we measured the frequency of H-2^b (donor C57BL/6)-positive cells within the T-cell, B-cell, granulocyte, and monocyte populations to assess peripheral blood chimerism. As expected due to the inability of untransduced HSCs to survive in extended culture and the anticipated 300-fold expansion of transduced HSCs, essentially all H-2^b-positive donor cells retained GFP expression (Fig. 2A). Across the range of NUPA10hd-LSK transplanted cells, the number of mice chimeric was found to be proportional to the number of cells injected (Fig. 2B). Strikingly even with a transplant dose as low as 1/100,000 of the culture, representing fewer than 0.03 starting HSCs or 9 HSCs after expansion, significant levels of chimerism were achieved in 1/5 mice. High levels of chimerism were achieved in the majority of mice with as little as 1/1,000th of the culture or 3 starting HSCs or 900 HSCs after expansion and in all mice with a maximum transplant does of 1/100th of a culture or the expanded progeny of only 30 starting HSCs. By comparison, chimerism in recipients of freshly sorted allogeneic LSK cells was only achieved in 3/5 mice at transplant dose of 3,000 (~100 HSCs). Furthermore, all chimeric mice presented high levels of chimerism for all four hematopoietic lineages tested, reflecting a very efficient and stable BM marrow reconstitution from donor mice (Fig. 2C). Importantly, no recipients that received NUPA10hd-transduced cells showed any evidence of myeloproliferative disorder or leukemia up to 3 months posttransplantation, consistent with earlier observations (13). Furthermore, the same recipients showed no obvious signs of GVHD, such as facial swelling or diarrhea. These results demonstrate that HSCs can be expanded several hundred fold and still sustain long-term donor-specific chimerism in mice bearing a fully mismatched MHC.

Figure 2.

LSK cells expressing NUPA10hd efficiently reconstitute allogenic mice in a long-term manner. (A–C) Blood cells were analyzed for Gr1, CD3, B220, Mac-1, and H-2^b. Recipient mice transplanted with LSK cells (untreated or infected with the NU-PA10hd construct) were bled and the frequency of donor reconstitution was measured by flow cytometry. (A) Dot plots show a nonchimeric (left plot) and a chimeric mouse (right plot). (B) Histogram shows the proportion of chimeric and nonchimeric mice. (C) Histogram shows the proportion of chimerism for B cells, T cells, granulocytes, and monocytes in chimeric mice, at 12 weeks after transplantation. Data are means ± SE of values of each blood cell subset from all chimeric mice of each group (N = 5).

Mice with High Level of Chimerism Are Tolerant for Muscle Engraftment

To determine whether recipients engrafted with NUPA10hd-expanded HSCs would tolerate subsequent transplants of therapeutic cells from the same source, we injected FACS-sorted myogenic progenitors (MPs) into the tibialis anterior (TA) muscles of mice that had received HSCs 12 weeks before. Upon transplantation, myogenic progenitors normally fuse with resident myofibers (9). Incorporation of immunologically mismatched MPs generally leads to destruction of the whole recipient fiber by the host immune system. Thus, we tested the ability of therapeutic C57BL/6 muscle cells to integrate and survive in NUPA10hd-LSK tolerized mice. The scheme used in these experiments is represented in Figure 3A.

Figure 3.

High level of chimerism in mice transplanted with NUPA10hd-transduced cells allows allogenic muscle cell engraftment. (A) Experimental design of MP transplantation. (B) Dot plots show the myogenic progenitor (MP) cell population sorted for intramuscular injection. (C) Muscle section containing engrafted GFP-positive cells from the donor (right) and Hoechst staining of the same section (left) (N = 5).

Skeletal muscle harvested from C57BL/6 mice ubiquitously expressing GFP was enzymatically dissociated, CD45^–, CD31^–, α7 integrin⁺ Sca-1^– MPs were purified as previously described (9) (Fig. 3B), and 3 × 10⁴ sorted cells were injected into the TA muscle of transplanted Balb/c mice as well as C57BL/6 control mice that had received syngenic BM. Four weeks after MP injection, limb muscles of recipients were analyzed for the presence of integrated GFP⁺ myofibers (Fig. 3C). All mice with a high level of chimerism accepted the muscle cell graft with no evidence of inflammatory cell infiltration (Table 1, Fig. 3C). In contrast, no GFP^– myofibers were found in nonchimeric mice or those with milder level of chimerism (Table 1). As a syngenic control group, Ly 5.1 C57BL/6 mice transplanted with 3,000 Ly 5.2 NUPA10hd-expressing LSK cells and displaying high levels of chimerism also received MPs by intramuscular injection. As expected, all mice within this group retained GFP myofibers (Table 1). As a control for myofiber rejection, C57BL/6 GFP⁺ MPs were injected in a group of Balb/c mice transplanted with 10⁶ Balb/c BM cells. Surprisingly in two independent experiments, one of four mice in this group was found to contain abundant positive GFP myofibers at the injection site (Table 1; data not shown). This might be due to immunosuppression caused by irradiation. Importantly, in order to ensure that detected GFP⁺ myofibers arose from fusion of injected GFP⁺ myoblasts and not of NUPA10hd-GFP-expressing HSCs, a group of Balb/c recipients was injected with 30,000 NUPA10hd-transduced LSK cells without receiving a following GFP⁺ myoblast injection. We did not observe any GFP⁺ myofiber in the chimeric mice from this group (data not shown).

Table 1.

Mice With High Level of Chimerism Accepted Muscle Graft

		Fraction of Culture/No. of Transplanted Cells	HSC Content as Starting Equivalents	Estimated HSC Transplanted	No. of Mice With Accepted Muscle Graft
Recipient Strain	Cell Type	Fraction of Culture/No. of Transplanted Cells	HSC Content as Starting Equivalents	Estimated HSC Transplanted	Chimeric Mice (for BM)	Nonchimeric Mice (for BM)
Balb/c	NUPA10hd-LSK (C57BL/6)	1/10⁶	0.003	0.9	na	0/4
Balb/c	NUPA10hd-LSK (C57BL/6)	1/10⁵	0.03	9	1/1	0/3
Balb/c	NUPA10hd-LSK (C57BL/6)	1/10⁴	0.3	90	1/2	0/3
Balb/c	NUPA10hd-LSK (C57BL/6)	1/10³	3	900	3/3	0/1
Balb/c	NUPA10hd-LSK (C57BL/6)	1/10²	30	9,000	5/5	na
Balb/c	fresh LSK (C57BL/6)	3,000	100	na	3/3	0/2
C57BL/6	fresh LSK (C57BL/6) (positive control)	300,000	10,000	na	5/5	na
Balb/c	total BM (Balb/c) (negative control)	10⁶	3,000	na	1/4	na

Positive control corresponds to group of C57BL/6 mice transplanted with syngenic C57BL/6 BM and negative control corresponds to Balb/c mice transplanted with syngenic Balb/c BM. na, not applicable.

Chimeric Mice Exhibit Functional T-Cell Tolerance

As a secondary assessment of immune tolerance induction, we performed a mixed lymphocyte reaction (MLR) assay, which involves a short-term coculture of bulk splenocytes recovered from transplanted animals and irradiated allogenic APCs. This simple assay reflects tolerance induction by asking whether BM recipient splenocytes are induced to proliferate in response to MHC-antigen determinants on allogenic APCs. Spleen cells (2 × 10⁵) from NUPA10hd-HSC transplanted mice were harvested 16 weeks after transplantation and cultured in the presence of 10⁴, 5 × 10⁴, or 10⁵ irradiated naive C57BL/6 spleen cells (stimulator cells, or APCs). Cell viability was confirmed by response to anti-CD3 (2C11) stimulation, and naive C57BL/6 and Balb/c responder groups were included to confirm that the assay was working properly (Fig. 4). From each of the three groups exhibiting both chimeric and nonchimeric mice (transplanted with 300, 3,000, and 30,000 NUPA10hd-transduced LSK cells, respectively), one chimeric and one nonchimeric mouse were selected to be tested by MLR. Proliferation of reconstituted Balb/c host T cells in response to C57BL/6 APC was found in all three cultures derived from the nonchimeric animals (Fig. 4), whereas cultures initiated from the spleen of mice demonstrating chimerism in circulating hematopoietic cells by FACS and tolerant to a muscle transplant did not produce T-cell proliferation in the MLR. These results suggest that clonal deletion occurred in the mice tolerant for satellite cell allograft.

Figure 4.

Chimeric mice exhibit immune tolerance. MLR (average cpm of triplicate cultures) of bulk host splenocytes in response to irradiated allogenic APCs. Responder cells (2 × 10⁵) from the spleen of NUPA10hd-LSK transplanted mice were cocultured with 10⁴, 5 × 10⁴, and 10⁵ stimulator APC from naive C57BL/6 mice, after which cell proliferation was determined by incorporation of [³H]TdR. Data in all panels are means ± SE of values from triplicate wells.

Discussion

Successful myoblast transplantations have been obtained in mice, monkeys, and even in DMD patients under tacrolimus immunosuppression (10,20,21). However, current long-term pharmacological immunosuppressive treatments required by these techniques result in numerous undesirable side effects. One promising alternative strategy to reduce graft rejection without the use of lifelong immunosuppression is the induction of lasting immune tolerance by establishment of mixed hematopoietic chimerism. One way to obtain sufficient chimerism to reliably induce tolerance is to apply significant myeloablation (25). However, this regime routinely used to allow allogenic marrow engraftment is so toxic that it is almost exclusively used to cure lymphomas and leukemias. Alternatively, large numbers of donor cells, usually higher than are clinically available, can be combined with milder myeloablative treatments to obtain sufficient chimerism (25). Different strategies have been attempted to increase HSC availability, including ex vivo expansion of BM or blood-mobilized HSCs. Unfortunately, expanded HSC seem to have a reduced ability to maintain long-term engraftment (27). Another attractive approach is to use the highly expandable ES cells as a source of donor cells for both HSCs and tissue-regenerating cells (e.g., muscle progenitors). However, ES cell-derived HSCs are extremely poor at engrafting adults. Although they have been able to efficiently repopulate the BM of irradiated recipients when transduced with HOXB4, their reconstitution of the lymphoid compartment is limited, possibly due to a direct effect of this transcription factor on hematopoietic cell differentiation (18). On the contrary, the homeodomain of another related HOX gene, HOXA10, when fused to NUP98 (NUPA10hd), has been shown to induce HSC expansion in vitro and in vivo (15,19). We demonstrate here that NUPA10hd transduced cells are fully capable of repopulating the BM of immunologically mismatched recipients, allowing the induction of immunological tolerance to myoblast transplantation. Future testing of NUPA10hd-infected ES cell-derived HSCs will be required in order to ascertain whether this transcription factor, similarly to what shown for HOXB4, can improve their engraftment efficiency.

In our study, all mice displaying a high level of chimerism were also found to be tolerant of muscle progenitor grafts that were immunologically matched to the donor HSCs. Measured levels of NUPA10hd-induced HSC expansion in vitro as well as the correlation between the numbers of HSCs transplanted and the level of detected chimerism suggests an important role of expanded HSCs in facilitating the engraftment following allogeneic transplant. Recent studies performed in an autologous setting and based on a novel purification strategy for in vitro expanded HSCs (manuscript in preparation) demonstrate that the efficiency of engraftment is directly proportional to the number of expanded HSCs. However, given the experimental design used in this study, we cannot exclude that, in addition to expanded HSCs, less primitive progenitors may also have contributed to the establishment of tolerance. Additionally, MLR assays suggested that graft-reactive T cells were deleted in chimeric mice, a process thought to yield the strongest form of tolerance (25). Therefore, our study highlights a method for obtaining large numbers of HSCs with similar tolerance-inducing properties as those obtained with extremely high (and thus clinically prohibitive) numbers of nonmanipulated HSCs. This strategy is of particular interest for the expansion of umbilical cord blood stem cells, which are available in limiting quantities but have promising properties for BM transplantation including less susceptibility to induce GVHD (6).

In order to improve our model, milder myeloablative conditioning needs to be tested, because irradiation is not routinely used in clinics to prepare the recipients for transplantation. Among the current options, a good alternative may include the use of busulfan combined with cylcophosphamide, which is currently the most widely used myeloablative regimen alternative to irradiation to treat hematologic disorders. Moreover, this bone marrow conditioning has shown promising results in terms of its ability to induce tolerance in mice (23). Furthermore, in order to induce a more efficient myoblast engraftment, additional approaches such as overexpression of follistatin could be combined to our strategy (3).

In conclusion, our study highlights the ability of NUPA10hd to increase the availability of HSCs for BM transplantation without affecting HSCs' property to induce chimerism and tolerization of muscle allograft in mismatched recipients. This strategy, combined with current clinical BM conditioning for allograft, would be a promising strategy in order to improve graft tolerance in a vast range of clinical situations.

Footnotes

Acknowledgments

We thank A. Johnson for help with flow cytometry, M. Williams for antibody production, and Y. Lai for technical help. This work was supported by a core grant from the NCE-Stem Cell Network to F.M.V.R., K.M.M., and R.K.H., CIHR grant MOP 160678 to F.M.V.R., and a grant from the Terry Fox Foundation to R.K.H. F.M.V.R. holds a Canada Research Chair in Regenerative Medicine. K.M.M. is a Michael Smith Foundation for Health Research Senior Scholar. J.L.B. is supported by postdoctoral fellowships from Multiple Sclerosis Society of Canada and Strategic Training Program in Transplantation (CIHR/UBC).

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NUP98-HOXA10hd-Expanded Hematopoietic Stem Cells Efficiently Reconstitute Bone Marrow of Mismatched Recipients and Induce Tolerance

Abstract

Keywords

Introduction

Materials and Methods

Retroviral Vectors

Animals

Antibodies and Staining Reagent for FACS Sorts

Purification of Lin–Sca+C-kit+ (LSK) Cells

Infection of LSK Cells with NUPA10hd Retrovirus

Transplant of Infected Cells and Flow Cytometry Analysis

Satellite Cell Sort and Transplantation

Tissue Analysis

Mixed Lymphocyte Reactions (MLR)

Results

Transplantation of BM Stem/Progenitor Cells Expressing NUPA10hd Induces Stable Hematopoietic Chimerism in Allogeneic Recipients

Mice with High Level of Chimerism Are Tolerant for Muscle Engraftment

Chimeric Mice Exhibit Functional T-Cell Tolerance

Discussion

Footnotes

Acknowledgments

References

Purification of Lin^–Sca⁺C-kit⁺ (LSK) Cells