Extensive Studies on Perfusion Method Plus Intra-Bone Marrow-Bone Marrow Transplantation Using Cynomolgus Monkeys

Authors


Abstract

The collection of bone marrow cells (BMCs) using a perfusion method has been advantageous not only because of the low contamination of BMCs with T cells from the peripheral blood but also the enrichment of stromal cells, which support hemopoiesis. Before the application of this new method to humans, its safety needed to be confirmed using cynomolgus monkeys. We therefore performed the perfusion method on more than 100 cynomolgus monkeys using the long bones (such as the humerus and femur) and also the iliac bones (for human application); in the more than 150 trials to date, there have been no accidental deaths. Furthermore, the technical safety of a new method for the intra-bone marrow (IBM) injection of BMCs (termed IBM-bone marrow transplantation) has also been confirmed using 30 monkeys.

Disclosure of potential conflicts of interest is found at the end of this article.

Introduction

Bone marrow transplantation (BMT) is one of the most powerful strategies for the treatment of various intractable diseases, including leukemia, aplastic anemia, congenital immunodeficiency, autoimmune diseases, and also for gene therapy and organ transplantation. In a series of experiments, we have found that the strategy of portal venous (PV) plus supplemental intravenous (IV) injections of donor whole bone marrow cells (BMCs) can induce persistent tolerance in chimeric-resistant MRL/lpr mice [1] and also in the skin allograft system [2, 3]. However, this method has demerits for human application; a laparoscope-guided injection of BMCs via the PV is necessary, and an additional IV injection is essential for obtaining a 100% success rate [1]. The tolerance induced by the PV injection of BMCs can be attributed to the donor-derived stromal cells being trapped in the liver; they facilitate the proliferation and differentiation of donor hemopoietic stem cells (HSCs) [4]. Based on these findings, we have established a new strategy for allogeneic BMT. Instead of the injection of BMCs via the PV or IV route, we injected them directly into the bone marrow cavity [5]. This method, termed intra-bone marrow-BMT (IBM-BMT), has an advantage for successful allogeneic BMT; allogeneic donor cells injected into the bone marrow cavity can effectively interact with donor-derived stromal cells, which support the proliferation, differentiation, and even maintenance of HSCs [6, 7], resulting in the earlier engraftment of hematolymphoid cells of donor-origin, in contrast to the conventional intravenous BMT (IV-BMT) [5]. In the case of IV-BMT, most BMCs (including HSCs and stromal cells) are trapped in the lung [8, [9]–10] and liver [11], where they are killed by radio-resistant host cells. In contrast, IBM-BMT can efficiently recruit donor HSCs and stromal cells into the bone marrow and can rapidly accelerate the proliferation of donor-derived HSCs while simultaneously retaining HSCs [5, 12, 13]. Using this new strategy, we have succeeded in treating intractable autoimmune diseases in chimeric-resistant MRL/lpr mice [5]. In addition, IBM-BMT can effectively induce donor-specific tolerance, which leads to the success of organ transplantation, including skin [14], pancreas [15], and hind limb transplantation [16]. Furthermore, it should be noted that, in IBM-BMT, donor cell engraftment can be achieved even with reduced radiation doses and without using any immunosuppressants [16].

Recently, we have also established a new method (perfusion method [PM]) for collecting BMCs from the long bones (the humerus, femur, and tibia) and also from the iliac bone using cynomolgus monkeys to minimize the contamination of BMCs with T cells from the peripheral blood [17, 18], resulting in the decreased frequency of acute graft-versus-host disease (GvHD) when allogeneic BMT is performed. Furthermore, the PM only requires two holes for the insertion of syringes in the long bones (or less than five holes in the iliac bone), thereby reducing the burden on bone marrow donors, compared with the conventional aspiration method (AM), in which BMCs have to be collected by multiple aspirations (>100 holes) from both the iliac bones under general anesthesia [19].

Therefore, the combination of the PM (for BMC collection) with IBM-BMT (for BMC injection) is thought to be an easy and effective method for achieving successful allogeneic BMT. Before the application of this combined method to humans, the technical safety of these methods needed to be confirmed using cynomolgus monkeys. We here show that this strategy (PM + IBM-BMT) is more safe and efficient than the conventional method (AM + IV-BMT).

Materials and Methods

Animals

Normal 2- to 4-year-old cynomolgus monkeys (2.7–3.7 kg) were obtained from CELESTE (Tokyo). The monkeys were free of intestinal parasites and seronegative for B virus, tuberculosis, herpes B virus, hepatitis A virus, and hepatitis B virus. All surgical procedures and postoperative care of animals were carried out in accordance with the guidelines of the National Institutes of Health for care and use of primates. The study protocol was approved (number 06-013) by the Animal Experimentation, Use, and Care Committee, Kansai Medical University.

Instrument for BMC Harvesting

The perfusion method is expected to be capable not only of harvesting large amounts of HSCs and stromal cells (including mesenchymal stem cells [MSCs]) but also of reducing the contamination of BMCs with T cells from the peripheral blood. In order to accomplish this, an instrument for BMC harvesting has been developed (JIMRO-TRANS; JIMRO Co. Ltd., Gunma, Japan, http://www.jimro.com). As shown in Figure 1, this bone marrow-harvesting needle has an inner needle with a drilling tip and helical groove. The rotation of the inner needle is transferred via a speed reduction mechanism to the mantle, which is prevented from rotating at the same speed as the inner needle by a co-rotation-prevention mechanism. The fine shards of bone generated by the rotation of the cutting edge of the mantle are collected onto the tip of the inner needle, transferred up the inner needle via the helical groove, and then discharged into the container in the upper portion. After drilling through the bone, the inner needle is withdrawn from the mantle, leaving the mantle still in place in the bone. This mechanism thus allows a hole to be drilled in bones such as the long bones as well as the iliac bone simply by rotating the bone marrow-harvesting needle.

Figure Figure 1..

Instrument for bone marrow cell harvesting. The bone marrow-harvesting needle has an inner needle with a drilling tip and helical groove. The rotation of the inner needle is transferred via a speed reduction mechanism to the mantle, which is prevented from rotating at the same speed as the inner needle by a co-rotation-prevention mechanism. The fine shards of bone generated by the rotation of the cutting edge of the mantle are collected onto the tip of the inner needle, transferred up the inner needle via the helical groove, and then discharged into the container in the upper portion. After drilling through the bone, the inner needle is withdrawn from the mantle, leaving the mantle still in place in the bone. This mechanism thus allows a hole to be drilled in the bones such as the long bones as well as the iliac bone simply by rotating the bone marrow-harvesting needle using an electric drill.

BMC Harvesting by PM

Cynomolgus monkeys were anesthetized using KETALAR (5 mg; Sankyo Co. Ltd., Tokyo, http://www.daiichisankyo.com), and an analgesic agent, PENTAGIN (Sankyo Co. Ltd.), was injected before the operation as previously described [17, 18]. The bone marrow fluid was then collected using the following procedures. A bone marrow puncture needle (consisting of an inner needle and outer sheath) was inserted at one end of a long bone such as the humerus or femur. In the case of collection of BMCs from the iliac bone, one bone marrow puncture needle was inserted into the end of the iliac crest and the other needle was inserted into the edge of the iliac crest. In some cases, a newly devised BMC harvesting set (Fig. 1) was used. After insertion was completed using this instrument connected to an electric drill, the inner needle was removed and a 30-ml syringe (code number SS-30ESZ; Terumo Co., Tokyo, http://www.terumo.co.jp/English), including 0.5 ml of heparin (10 U/ml in saline; Novo Nordisk, Copenhagen, Denmark, http://www.novonordisk.com), was connected to the outer sheath, and the same procedure was performed at the other end of the long bone or the iliac bone. A 30-ml syringe containing 30 ml of saline was connected to the outer sheath, and the saline was then pushed gently from the syringe into the medullary cavity to flush out the bone marrow (Fig. 2). The BMCs were collected into the syringe (0.5 ml of heparin: 10 U/ml in saline) that had been connected at the other end of the long bone or the iliac bone. In some experiments, BMCs were collected from the long bone or the iliac bone by the conventional AM using a bone marrow puncture needle.

Figure Figure 2..

Perfusion method. Using the instrument (Fig. 1) or commercially available bone marrow puncture needles, one bone marrow puncture needle was inserted into the end of the iliac crest and the other needle was inserted into the edge of the iliac crest. After the insertion was completed, the inner needle was removed, and a 30-ml syringe, including 0.5 ml of heparin (10 U/ml in saline), was connected to the outer sheath. A 30-ml syringe containing 30 ml of saline was connected to the outer sheath, and the saline was then pushed gently from the syringe into the medullary cavity to flush out the bone marrow.

Preparation of BMCs

BMCs that had been harvested by the PM were centrifuged and suspended at a concentration of 5 × 108 cells per milliliter to minimize the volume for injection into the bone marrow cavity. In some experiments, the BMCs were placed on 15 ml of Lymphoprep density solution (1.077 g/ml; Nycomed, Oslo, Norway, http://www.nycomed.com/en/menu). After centrifugation for 30 minutes at 2,000 rpm at room temperature, the BMCs (depleted of red blood cells) were collected from the defined layer at the interface and adjusted to 5 × 108 cells per milliliter.

Procedure for IBM-BMT

Recipient cynomolgus monkeys received the whole BMCs by IBM-BMT. IBM-BMT was performed as follows. Each cynomolgus monkey was anesthetized using KETALAR, and an analgesic agent (PENTAGIN) was then injected. The bone marrow puncture needle (consisting of the inner needle and outer sheath) or a BMC-harvesting set (Fig. 1) was inserted into the long bones such as the humerus or tibia. After the insertion was completed, 1 ml of BMCs (adjusted to 1–5 × 108 per milliliter) were slowly injected (30 μl/second) into the bone marrow cavity of both the humeri or both the tibiae. Bone wax was used to prevent the BMCs spilling from the bone cavity.

Analyses of Cell Surface Antigens

Cell surface antigens on the peripheral blood mononuclear cells (PBMNCs) and BMCs were determined using phycoerythrin-coupled monoclonal antibodies (mAbs) against human CD4 and CD8 (Exalpha, Boston, http://www.exalpha.com). These mAbs were previously examined for their cross-reactivities to the molecules expressed on the cells of cynomolgus monkeys. Flow cytometric analyses were performed using a FACScan (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).

Colony-Forming Assays

The colony-forming ability of BMCs (colony-forming unit in culture [CFU-C]) was assayed as described previously [16]. Briefly, BMCs (104 cells per well) were plated in 12-well plates (MP Biomedicals, Solon, OH, http://www.mpbio.com) in a volume of 1 ml of MethoCult GF H4434 (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) consisting of optimal concentrations of cytokines (recombinant human stem cell factor [SCF], erythropoietin [EPO], interleukin [IL]-3, granulocyte macrophage–colony-stimulating factor [GM-CSF], and granulocyte–colony-stimulating factor [G-CSF]), 30% fetal bovine serum (FBS), 1% bovine serum albumin, 2 mM l-glutamine, 10−4 M 2-mercaptoethanol, and 0.9% methyl cellulose. Fourteen days later, the CFU-C counts were determined.

Furthermore, to examine the activity of BMCs to generate stromal cells, BMCs (3 × 103, 1 × 104, and 3 × 104 cells per well) were plated and cultured for 2 weeks in 96-well plates in a volume of 100 ml of Dulbecco's modified Eagle's medium with 10% FBS. The wells where stromal cells were observed were counted as positive wells.

Results

Number of Sites at Which BMCs Were Harvested by PM

The bone marrow fluid was collected from the long bones using the instrument shown in Figure 1. The first needle was connected to a 30-ml syringe containing 0.5 ml of heparin (10 U/ml in saline), and the other needle was connected to a syringe containing 30 ml of saline. The saline was pushed gently from the syringe into the medullary cavity to flush out the bone marrow.

The perfusion method using the iliac bone was performed as shown in Figure 2. One needle was inserted into the abdominal iliac spine, and the other needle was inserted into the dorsal iliac spine. The first needle was connected to a syringe containing 0.5 ml of heparin, and the other needle was connected to a syringe containing 30 ml of saline. The bone marrow fluid was collected using a technique similar to that used for the long bones.

The sites from which cells were harvested using the PM are summarized in Table 1. We carried out extensive experiments from 2004–2006 and confirmed that this PM is safe even if BMCs are collected from various bones, including the iliac bones.

Table Table 1.. Number of sites at which bone marrow cells were harvested by perfusion method
original image

Differences in Quantities and Qualities of BMCs Harvested by PM and AM

The average number of BMCs harvested by the PM was ∼15 × 107 cells from the humerus, ∼8 × 107 cells from the femur, and ∼9 × 107 cells from the iliac bone (Table 2). Thus, approximately 3 × 108 BMCs, in total, were obtained from one cynomolgus monkey, which is sufficient to achieve IBM-BMT of recipient cynomolgus monkeys weighing ∼3 kg (108 BMCs per kilogram). Furthermore, the PM has another advantage: BMCs were collected from these sites within 15 minutes on average, and the whole operation could be achieved within 60 minutes, suggesting that the PM presents a substantially reduced burden on the BM donors compared with the conventional AM.

Table Table 2.. Analyses of CD4+ and CD8+ cells
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It is well known that a significant contamination of BMCs with T cells from the peripheral blood is often observed when BMCs are obtained using the conventional AM. As shown in Table 2, more than 40% of T cells (CD4+ and CD8+ cells in a lymphocyte gate) were observed in the PBMNCs as a positive control, and more than 20% of T cells in the BMCs collected from the iliac bone by the conventional AM. In contrast, less than 10% of T cells were present in the BMC preparation collected by the PM from the long bones and iliac bones (p < .01, PM vs. AM). Therefore, there was significantly less contamination of BMCs with T cells from the peripheral blood when the BMCs were collected using the PM.

In vitro CFU-C assays were next carried out to examine the progenitor cell activity in the BMCs collected using the PM and the AM. The BMCs harvested from the humerus or iliac bone using the PM or AM were cultured in methylcellulose containing a combination of cytokines essential for hemopoiesis (SCF, EPO, IL-3, GM-CSF, and G-CSF). The BMCs collected using the PM generated a significantly higher number of CFU-C than those harvested using the conventional AM when assayed 14 days later, as shown in Figure 3. This indicates that the frequency of progenitor cells is higher in BMCs collected using the PM than the AM.

Figure Figure 3..

Measurement of CFU-C. The number of CFU-C of BMCs was measured in the culture containing a mixture of optimal concentrations of hemopoietic cytokines. BMCs (104 cells per well) were plated in 12-well plates consisting of recombinant human stem cell factor, erythropoietin, interleukin-3, granulocyte macrophage-colony-stimulating factor, and granulocyte-colony-stimulating factor, 30% fetal bovine serum, 1% bovine serum albumin, 2 mM l-glutamine, 10−4 M 2-mercaptoethanol, and 0.9% methyl cellulose. Fourteen days later, the CFU-C counts were determined. Columns and bars are means ± SD of five monkeys. Abbreviations: AM, aspiration method; BMCs, bone marrow cells; CFU-C, colony-forming unit in culture; PBMNCs, peripheral blood mononuclear cells; PM, perfusion method.

Furthermore, the ability to generate stromal cells (colony-forming unit–fibroblasts) was compared between the BMCs collected using the PM and the AM. The BMCs collected using the AM showed a slightly greater ability to generate stromal cells than those collected using the PM, although the difference was not significant (Table 3), indicating that stem cells (including MSCs) can be harvested using either the PM or the AM.

Table Table 3.. Percentages of wells with colony-forming unit–fibroblasts
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Safety of IBM-BMT

BMCs collected from the PM were transplanted directly into the bone marrow cavities of the long bones of recipient monkeys that had been irradiated. We performed 30 experiments and 30 trials of IBM-BMT in total. IBM-BMT was performed on both right and left humeri rather than the tibiae in the case of cynomolgus monkeys, since the monkeys would otherwise scratch the wound after the operation. The average number of BMCs transplanted by IBM-BMT was 1.88 ± 0.84 × 108 cells per kilogram, and no accidents occurred during the operation. These findings indicate that both the PM and IBM-BMT are safe and can be applied to humans.

Discussion

IBM-BMT has become an established method and has already been applied to humans [20, [21], [22], [23], [24], [25]–26]; drugs in fluids and/or blood have been administered intraosseously (i.o.) to critically ill patients. Furthermore, i.o. infusion (corresponding to IBM-BMT) has been compared with IV infusion in human allogeneic (allo) BMT, and it has been concluded that allo BMT can be safely performed by i.o. infusion [24]. The instances of acute and chronic GvHD, transplantation-related mortality, and survival rates were similar. However, Hagglund et al. aspirated donor BMCs from the iliac bones and infused a large volume of these BMCs into the iliac bones but not the long bones of the recipients. It has recently been reported that IBM-BMT is superior to IV-BMT in severe combined immunodeficient mice reconstituted with human cells [27, [28], [29]–30]. However, we have found that the effectiveness of IBM-BMT (i.o. infusion) is partially dependent on the volume to be infused into the bone marrow cavity [27]; a small volume (highly concentrated, 10∼20 × 108 cells per milliliter) of the infused BMCs (<10 μl in the case of murine IBM-BMT) can enhance the retention of BMCs (particularly stromal cells and HSCs) inside the bone marrow, resulting in the early engraftment of allogeneic or even xenogeneic (human) HSCs.

In our series of experiments, we have shown that IBM-BMT is, so far, the best strategy for allo BMT for the following reasons: (a) no GvHD develops even if T cells are not depleted from the bone marrow, since donor stromal cells produce immunosuppressive cytokines, such as hepatocyte growth factor and transforming growth factor-β, resulting in the prevention of GvHD in mice [31]; (b) hemopoietic recovery is rapid [5, 32]; and (c) the restoration of T-cell functions is complete even in donor-recipient combinations across the major histocompatibility complex barriers [5, 14, 1516]. We have also shown that the PM has the substantial advantages that no GvHD develops, since the BMCs thus collected include less than 10% T cells, and that a large number of BMCs can be collected quickly [17, 18]. The safety of this method has been confirmed in the present study. We therefore believe that the combination of the PM and IBM-BMT (i.o. infusion of a highly concentrated and small volume of BMCs) will become a powerful new strategy for not only allo BMT but also organ transplantation in conjunction with IBM-BMT.

Indeed, we have very recently carried out “PM + IBM-BMT” in a patient with β-thalassemia major [25]. The recipient was a 6-year-old girl, and the donor was her 37-year-old father (4/6 human leukocyte antigen matched). First, we carried out the PM to collect BMCs from her father. It took a total of approximately 1 hour to collect the BMCs. The BMCs contained 6% of CD3+ T cells and 1% of CD34+ cells. The donor could walk the next day without pain. IBM injection of the whole BMCs (1 × 108 per milliliter) into both tibias (10 ml each) of the recipient was carried out within 30 minutes. The white blood cell counts of the patient gradually increased to over 1,500 per microliter by day 47 and continued to increase, reaching the highest level (8,600 per microliter) on day +55. Fluorescence in situ hybridization data on day +33 showed that 98% of the peripheral blood cells were from the donor. Notably, there were no clinical symptoms of GvHD. Thus, the new BMT method (PM + IBM-BMT) was successfully carried out, but, on day +56, the patient regrettably died of asphyxia resulting from sticky sputum. There was no evidence of infection (in the lung or liver) or GvHD (in the skin) by necropsy [25].

We are now in the process of establishing an exact protocol (including conditioning regimens) for clinical applications using cynomolgus monkeys (manuscript in preparation). We have also started a “phase I study” to confirm the safety of the PM using poor mobilizer patients in malignant lymphomas (manuscript in preparation).

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

Acknowledgements

The authors thank Y. Tokuyama, K. Hayashi, and A. Kitajima for their expert technical assistance and Hilary Eastwick-Field, Brian O'Flaherty, and K. Ando for their help in the preparation of the manuscript. This work was supported by a grant from Haiteku Research Center of the Ministry of Education, a grant from the Millennium program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from the Science Frontier program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from the 21st Century Center of Excellence (COE) program of the Ministry of Education, Culture, Sports, Science and Technology, and also a grant from the Department of Transplantation for Regeneration Therapy (sponsored by Otsuka Pharmaceutical Company, Ltd.), a grant from Molecular Medical Science Institute (Otsuka Pharmaceutical Co., Ltd.), and a grant from Japan Immunoresearch Laboratories Co. (JIMRO).

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