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. 2013 Apr 11:11:96.
doi: 10.1186/1479-5876-11-96.

Intra-osseous injection of donor mesenchymal stem cell (MSC) into the bone marrow in living donor kidney transplantation; a pilot study

Hyunah Lee  1 , Jae Berm Park, Sanghoon Lee, Soyoung Baek, HyunSoo Kim, Sung Joo Kim
Affiliations

Intra-osseous injection of donor mesenchymal stem cell (MSC) into the bone marrow in living donor kidney transplantation; a pilot study

Hyunah Lee et al. J Transl Med. .

Abstract

Background: Mesenchymal stem cells (MSCs) are multi-potent non-hematopoietic progenitor cells possessing an immune-regulatory function, with suppression of proliferation of activated lymphocytes. In this study, adult living donor kidney transplantation (LDKT) recipients were given MSCs derived from the donor bone marrow to evaluate the safety and the feasibility of immunological changes related to the intra-osseous injection of MSC into the bone marrow.

Methods: MSCs were derived from negative HLA cross-match donors. Donor bone marrow was harvested 5 weeks prior to KT. At the time of transplantation, 1 x 106 cell/kg of donor MSC was directly injected into the bone marrow of the recipient's right iliac bone. Patients' clinical outcomes, presence of mixed chimerism by short tandem repeat polymerase chain reaction, analysis of plasma FoxP3 mRNA and cytokine level, and mixed lymphocyte reaction (MLR) were performed.

Results: Seven patients enrolled in this study and received donor MSC injections simultaneously with LDKT. The median age of recipients was 36 years (32 ~ 48). The number of HLA mismatches was 3 or less in 5 and more than 3 in 2. No local complications or adverse events such as hypersensitivity occurred during or after the injection of donor MSC. There was no graft failure, but the biopsy-proven acute rejections were observed in 3 recipients during the follow-up period controlled well with steroid pulse therapy (SPT). The last serum creatinine was a median of 1.23 mg/dL (0.83 ~ 2.07). Mixed chimerism was not detected in the peripheral blood of the recipients at 1 and 8 week of post-transplantation. Donor-specific lymphocyte or T cell proliferation and Treg priming responses were observed in some patients. Plasma level of IL-10, a known mediator of MSC-induced immune suppression, increased in the patients with Treg induction.

Conclusion: Donor MSC injection into the iliac bone at the time of KT was feasible and safe. A possible correlation was observed between the induction of inhibitory immune responses and the clinical outcome in the MSC-kidney transplanted patients. Further research will be performed to evaluate the efficacy of MSC injection for the induction of mixed chimerism and subsequent immune tolerance in KT.

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Figures

Figure 1
Figure 1
Level of FoxP3 mRNA. Quantitative real time PCR with recipient urine pellets was performed and analyzed FoxP3 mRNA expression to estimate the induction of regulatory T cells. Following the methods in the Materials & Methods section, Foxp3 mRNA level in the MSC-kidney transplantation patients was measured. Patients’ urine was collected from day 0 and days 7, 30, 90, 180 and 365 after the transplantation. Foxp3 mRNA level was compared to baseline level (= 1).
Figure 2
Figure 2
Donor-Specific Lymphocyte Proliferation (mixed lymphocyte reaction). Heparinized blood sample of recipient was collected before (day-35) and after (day + 30) the MSC-kidney transplantation. The recipients lymphocytes (responder) were co-cultured with irradiated donor lymphocytes (stimulator) for 4 days. Donor-specific recipients’ lymphocyte proliferation was determined by 3H-thymidine incorporation using a liquid scintillation counter (Beckman LS 6500; Beckman Instruments Inc., Fullerton, CA, USA). Proliferative response was represented by DPM. Plus signs indicate statistically significant induction of proliferative response (p < 0.05) compared to D-35 baseline activity. Asterisks indicate the statistical significance of decreased proliferative response compared to D-35 baseline activity (p < 0.05).
Figure 3
Figure 3
Donor-Specific T cell Proliferation (mixed lymphocyte reaction). Co-culture of recipient’s blood lymphocytes and irradiated donor lymphocytes was stimulated by the T cell mitogen, ConA. Same experimental procedure was performed as MLR for lymphocyte proliferation analysis. Proliferative response was represented by DPM. Plus signs indicate the statistically significant induction of proliferative response (p < 0.05) compared to D-35 baseline activity. Asterisks indicate the statistical significance of decreased proliferative response compared to D-35 baseline activity (p < 0.05).
Figure 4
Figure 4
IL-10 secretion. Alteration in blood circulating concentrations of IL-10 was measured by ELISA. Heparinized blood sample of donor or recipient was collected before (day-35) and at various times after (day + 4, 7, 30, 90, 180) the MSC-kidney transplantation. Plasma was separated and stored at −70°C until the ELISA was performed using commercial kit from OptEIA™ (e-bioscience, San Jose, CA, USA). Asterisks indicate the statistical significance of cytokine secretion compared to D-35 baseline activity (p < 0.05).
Figure 5
Figure 5
IL-6 secretion. Alteration in blood circulating concentrations of IL-6 was measured by ELISA. Heparinized blood sample of donor or recipient was collected before (day-35) and at various time after (day + 4, 7, 30, 90, 180) the MSC-kidney transplantation. Plasma was separated and stored at −70°C until the ELISA was performed using commercial kit from OptEIA™ (e-bioscience, San Jose, CA, USA). Asterisks indicate the statistical significance of cytokine secretion compared to D-35 baseline activity (p < 0.05).
Figure 6
Figure 6
IFN-γ secretion. Alteration in blood circulating concentrations of IFN-γ was measured by ELISA. Heparinized blood sample of donor or recipient was collected before (day-35) and at various time after (day + 4, 7, 30, 90, 180) the MSC-kidney transplantation. Plasma was separated and stored at −70°C until the ELISA was performed using commercial kit from OptEIA™ (e-bioscience, San Jose, CA, USA). Asterisks indicate the statistical significance of cytokine secretion compared to D-35 baseline activity (p < 0.05).
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