Stable Engraftment Research Articles

Properties of Porcine and Recombinant Human Collagen Matrices for Optically Clear Tissue Engineering Applications

Porcine and recombinant human atelocollagen I solutions were cross-linked with a water soluble carbodiimide at various stoichiometries and collagen concentrations (5-20 w/w %). The resulting hydrogels were clear and, when used as cell growth matrices, allowed cell and nerve visualization in vitro and in vivo. We have previously reported that, after six months of implantation in pigs' and rabbits' corneas, these robust hydrogels allowed regeneration of host cells and nerves to give optically clear corneas with no detected loss in thickness, indicating stable engraftment. Here, the biocompatible hydrogel formulations leading to this novel in vivo performance were characterized for amine consumption, gel hydration, thermal properties, optical clarity, refractive index, nutrient diffusion, biodegradation, tensile measurements, and average pore diameters. Gels with excellent in vitro (epithelial overgrowth, neurite penetration) and in vivo performance (clarity, touch sensitivity regeneration) had 4-11 nm pores, yet had glucose and albumin diffusive coefficients similar to mammalian corneas and allowed neurite extension through the gels.

Stable Engraftment after a Conditioning Regimen with Fludarabine and Melphalan for Bone Marrow Transplantation from an Unrelated Donor

Graft failure and nonrelapse mortality (NRM) are major obstacles after the first unrelated-donor bone marrow transplantation (UD-BMT) with reduced-intensity conditioning. We evaluated UD-BMT with fludarabine (5 x 25 mg/m2) and melphalan (2 x 90 mg/m2) treatment combined with short-term methotrexate and tacrolimus (n = 20) or cyclosporine (n = 2) therapy for 22 patients with hematologic malignancies who were ineligible for conventional conditioning. Only 9 patients were in remission at transplantation. Seventeen patients underwent HLA-matched or DRB1 allele-mismatched transplantation, and 5 patients underwent HLA-A allele-mismatched or serologically HLA-DR-mismatched transplantation. Regimen-related toxicities were tolerable, although transient oral mucositis, hepatobiliary enzyme elevation, and diarrhea were observed frequently. All evaluable patients achieved sustained neutrophil engraftment, and all patients tested showed complete donor chimerism on day 28. With a median follow-up of 16 months, NRM and overall survival rates at 1 year were 19% and 81%, respectively, among the patients who underwent HLA-matched or DRB1 allele-mismatched transplantation. Acute graft-versus-host disease (GVHD) of grades II to IV occurred in 26% of the patients. The cumulative incidence of chronic GVHD was 44%. Despite the small number of patients and the short follow-up period, this reduced-intensity regimen enabled satisfactory engraftment and achievement of rapid complete donor chimerism with tolerable toxicities in the patients, including those who underwent HLA-mismatched UD-BMT.

Hematopoietic Engraftment of Human Embryonic Stem Cell‐Derived Cells Is Regulated by Recipient Innate Immunity

Human embryonic stem cells (hESCs) provide an important means to characterize early stages of hematopoietic development. However, the in vivo potential of hESC-derived hematopoietic cells has not been well defined. We demonstrate that hESC-derived cells are capable of long-term hematopoietic engraftment when transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Human CD45(+) and CD34(+) cells are identified in the mouse bone marrow (BM) more than 3 months after injection of hESCs that were allowed to differentiate on S17 stromal cells for 7-24 days. Secondary engraftment studies further confirm long-term repopulating cells derived from hESCs. We also evaluated two mechanisms that may inhibit engraftment: host immunity and requirement for homing to BM. Treatment with anti-ASGM1 antiserum that primarily acts by depletion of natural killer cells in transplanted mice leads to improved engraftment, likely due to low levels of HLA class I expressed on hESCs and CD34(+) cells derived from hESCs. Intra-BM injection also provided stable engraftment, with hematopoietic cells identified in both the injected and contra-lateral femur. Importantly, no teratomas are evident in animals injected with differentiated hESCs. These results demonstrate that SCID-repopulating cells, a close surrogate for hematopoietic stem cells, can be derived from hESCs. Moreover, both adaptive and innate immune effector cells may be barriers to engraftment of these cells.

G-CSF(filgrastim) on day −1 (day before stem cell infusion) provides stable engraftment and good progression free and overall survival (PFS and OS) in hematopoeitic stem cell transplantation (HSCT)-Tata Memorial Centre experience

Background: Recombinant human G-CSF hastens neutrophil recovery following hematopoietic stem cell transplantation (HSCT), thus accelerating engraftment.The optimal timing for use of G-CSF in conjunction to HSCT needs to be established.We present our data on effects of adding an extra dose of G-CSF (filgrastim 5 micro gm/kg) on day -1 of HSCT. Patients and Methods: Forty three consecutive patients who underwent HSCT in one year period were given G-CSF on day +1 (day before stem cell infusion) in addition to the regular doses from day +1 till engraftment.

AMD3100 + G-CSF improves hematopoietic progenitor cell (HPC) collection in patients with hodgkin’s disease (HD)

Autologous stem cell transplantation is indicated for patients with HD who have primary refractory disease or who relapse after a first remission. For these patients, as for other patients undergoing autologous transplantation, the number of CD34+ cells infused is a reliable predictor of neutrophil and platelet engraftment, and doses ≥ 5 x 10 6 CD34+ cells/kg are associated with faster count recovery. However, among the 98 patients with HD who have undergone G-CSF-alone mobilization at our institution in the past five years, 22% did not achieve a minimum HPC collection of 2 x 10 6 CD34+ cells/kg in ≤ 5 apheresis procedures, and only 15% achieved a collection ≥ 5 x 10 6 CD34+ cells/kg. AMD3100 mobilizes HPCs by reversibly inhibiting the interaction of CXCR4 and SDF-1α. It has been shown to improve HPC mobilization in patients with multiple myeloma and non-Hodgkin’s lymphoma. Here we present results for the first ten HD patients treated with a mobilization regimen of AMD3100 + G-CSF. To date, ten patients with relapsed (8) or refractory (2) HD have been mobilized with G-CSF (10 ug/kg/d) + AMD3100 (240 ug/kg/d) beginning on day 4. Apheresis was performed 11 hours after each AMD3100 dose. The first dose of AMD3100 produced a median (range) 3.0 (1.9–5.1) fold increase in the number of circulating CD34+ cells. Six patients (60%) achieved a collection of ≥ 5 x 10 6 CD34+ cells/kg, and all patients collected > 2 x 10 6 CD34+ cells/kg (range, 3.6–9.4 x 10 6 CD34+ cells/kg). The median (range) number of apheresis procedures performed per patient was 2 (1–4). No grade II-IV adverse events were ascribed to AMD3100. Eight patients have been transplanted with G-CSF + AMD3100 mobilized cells. All have had prompt and stable engraftment, with median neutrophil recovery at day +9 (9–11) and median platelet recovery at day +20 (15–23). Two patients had very early engraftment, with absolute neutrophil count greater than 100 on day +7. We conclude that AMD3100 + G-CSF is a well-tolerated and effective mobilization regimen in patients with HD. All patients (100%, 95% CI 69%-100%) mobilized with AMD3100 + G-CSF achieved the minimum collection of 2 x 10 6 CD34+ cells/kg, and a significantly higher proportion of patients (60%, 95% CI 26%–88%) achieved the goal collection of ≥ 5 x 10 6 CD34+ cells/kg than did the historical controls (15%). Importantly, the median collection in the first two days of pheresis was 5.4 x 10 6 CD34+ cells/kg, which is significantly better than historical controls, who collected a median 3.0 x 10 6 CD34+ cells/kg in the first two days of pheresis (p=0.014). Our results demonstrate that the mobilization regimen of AMD3100 + G-CSF can improve the number of HPCs collected and decrease the number of days of pheresis in HD patients. This regimen will be pursued further in this patient population.

Stable donor engraftment following reduced intensity hematopoietic cell transplantation for sickle cell disease

Stable donor engraftment following reduced intensity hematopoietic cell transplantation for sickle cell disease

Fludarabine phosphate and melphalan: a reduced intensity conditioning regimen suitable for allogeneic transplantation that maintains the graft versus malignancy effect

Reduced intensity conditioning (RIC) for allogeneic stem cell transplantation allows stable donor cell engraftment with the maintenance of a graft versus malignancy effect. Many different regimens exist employing various combinations of chemotherapy, radiotherapy and T-cell depletion. We examined the role of non-T-cell depleted RIC regimens in 56 patients with haematological malignancies. Patients received fludarabine phosphate for 5 days (30 mg/m2 in 35 patients, 25 mg/m2 in 21 patients) and melphalan for 1 day (140 mg/m2 in 36 patients, 100 mg/m2 in 20 patients). Immunosuppression was with CyA alone in 33 patients and CyA/MTX in 23 patients. Twenty-four of the 26 patients with chimerism data showed >95% donor chimerism at 3 months post transplant. aGVHD occurred in 18% of patients receiving CyA/MTX compared to 53% of patients receiving CyA. The 100-day mortality rate was 0.16 (95%CI 0.08-0.28) and 1-year nonrelapse mortality was 0.24 (95%CI 0.13-0.38). Thirty-three patients remained alive and in CR at a median of 19 months post transplant (range 3-38 months). We have shown that patients transplanted with fludarabine phosphate, melphalan 100 mg/m2 and with CyA/MTX as post transplant immunosuppression can achieve good disease control with an acceptable level of toxicity. Further studies are required to confirm these findings.

Reduced-intensity Umbilical Cord Blood Transplantation in Adult Acute Myeloid Leukemia: Report of Four Cases

The outcome after unrelated cord blood transplantation (CBT) is similar to that of matched unrelated bone marrow transplantation in children, and the results of CBT in adult patients has recently shown improvement. In addition, the use of reduced-intensity conditioning regimens for CBT has shown stable engraftment and reduced treatment-related mortality (TRM). From May 2005 to Jan 2006, four adult patients with acute myelogenous leukemia were treated with CBT after reduced-intensity conditioning at our hospital. The mean age of patients was 53.8 yrs, and all patients received 2 HLA antigen mismatched single unit cord blood. The infused mean cell dose was 2.85×107/kg for total nucleated cells and 0.72×105/kg for CD34+ cells. All patients had engraftment. The mean number of days to WBC and platelet engraftment was D+20.3 and D+60.3, respectively. There was no TRM within 100 days after transplantation. At the last follow up, three of the four patients were alive. One patient transplanted in first complete remisson is alive in remission at day 413, but the other patients transplanted in advanced disease all relapsed. Reduced-intensity CBT is a feasible approach in selected adult patients with acute myeloid leukemia.

Embryonic stem cell-derived hematopoietic stem cells

Despite two decades of studies documenting the in vitro blood-forming potential of murine embryonic stem cells (ESCs), achieving stable long-term blood engraftment of ESC-derived hematopoietic stem cells in irradiated mice has proven difficult. We have exploited the Cdx-Hox pathway, a genetic program important for blood development, to enhance the differentiation of ESCs along the hematopoietic lineage. Using an embryonic stem cell line engineered with tetracycline-inducible Cdx4, we demonstrate that ectopic Cdx4 expression promotes hematopoietic mesoderm specification, increases hematopoietic progenitor formation, and, together with HoxB4, enhances multilineage hematopoietic engraftment of lethally irradiated adult mice. Clonal analysis of retroviral integration sites confirms a common stem cell origin of lymphoid and myeloid populations in engrafted primary and secondary mice. These data document the cardinal stem cell features of self-renewal and multilineage differentiation of ESC-derived hematopoietic stem cells.

Monitoring Graft Function after First Allogeneic Transplant for Mucopolysaccharidosis-IH: Patterns of Rejection and Outcome of Second Allograft Procedure.

Mucopolysaccharidosis type I (MPS-I [Hurler's syndrome]) is an autosomal recessive condition caused by a deficiency of alpha-L-iduronidase. We report single centre 20 year experience of outcomes of BMT in MPS-IH. As there is no consensus about graft monitoring and the indication for second transplant, we describe the monitoring of graft function, indications for and management of second BMT in this institution.Data was collected from 43 patients receiving 61 transplants who underwent allogeneic transplantation at the Royal Manchester Children's Hospital between 1985 and 2005. The median age at first transplant was 15 months (range 7– 44), and of these 23 were male and 20 were female. 24 of the first transplants were from an unrelated donor. Graft function was monitored prospectively in each transplant using enzyme levels, urinary GAGs and, for the last 33 transplants, VNTR analysis.Almost half of the 61 transplants carried out since 1985 have been done in the last five years. Of the 43 patients transplanted, 19 survived with stable engraftment, 16 patients rejected their first grafts and all 16 of these patients went on to receive a second transplant. GVHD occurred in 16 of 28 patients who received only 1 transplant. Of the 16 who had 2 procedures, 6 had GVHD after their first transplant and 8 after their second. 8 patients died and all deaths were due to transplant-related complications (1 EBV/LPD, 1 CMV, 4 Adenovirus, 2 GVHD). 24 patients received unrelated donor grafts. 16 of these MUD transplants were carried out before 2002 and 8 since 2002. 6 of the 8 deaths were in patients who received unrelated donor grafts and all 6 of these were pre 2002. This improvement in mortality is significant (Fisher's exact test 2-sided p= 0.04).45 of the 61 transplants were carried out after 1995 and these patients all had regular VNTR monitoring of their graft function. From these results 4 groups of graft status are identified: full engraftment, stable mixed chimerism, immediate rejection, early autologous reconstitution. 23 patients remain fully engrafted at 100% VNTR levels. 16 of these were after first transplant and 7 following their second transplant. 4 patients have stable mixed chimerism (2 after 1st transplant and 2 after 2nd). Although the VNTR levels are now not 100% in this group, they remained at 100% until at least 70 days post transplant. They reached a state of stable mixed chimerism at a mean of 203 days post transplant (range 133–280). The final level of mixed chimerism reached was a mean of 59% donor. 3 patients immediately rejected their graft with VNTR levels falling to 0% within 28 days of transplantation. 8 patients had early autologous reconstitution requiring a second transplant procedure once their VNTR levels fell to 20%, and in these there was early mixed chimerism (within 60 days after graft).All 16 patients who rejected their first graft went on to have a second transplant when their VNTR levels fell to<20%. 13 of these survived with stable engraftment and 3 died. The donor was the same in all but one case, and conditioning was usually of reduced intensity.SummaryGraft rejection is not uncommon after allogeneic BMT for MPS-1H and can be detected using VNTR, enzyme and urinary GAGs. Where chimerism is detected earlier than 60 days the graft is subsequently lost, whereas later mixed chimerism usually stabilises at adequate levels. Second transplant is effective and safe using the same donor.

Postgrafting Immune Suppression Combined with Nonmyeloablative Conditioning for Transplantation of HLA-Identical Hematopoietic Cell Grafts: Results of a Phase I Study for Treatment of Immunodeficiency Disorders.

Myeloablative conditioning regimens may cause significant life threatening transplant related toxicities in patients with immunodeficiency disorders, particularly in patients with co-morbid conditions. This current study was designed to determine whether an immunosuppressive nonmyeloablative regimen would safely enable stable donor engraftment of hematopoietic cells in patients with immunodeficiency disorders who were ineligible for myeloablative conditioning regimens because of life-threatening infections or organ dysfunction. Fourteen patients with severe combined immunodeficiency disorder (SCID; n=3), common variable immunodeficiency (CVID; n=1), T-cell immunodeficiency disorder (TCD; n=3), Wiskott-Aldrich syndrome (WAS; n=2), CD40 Ligand deficiency (CD40LD; n=2), immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX; n=1), X-linked agammaglobulinemia (XLA; n=1) and chronic granulomatous disease (CGD; n=1) received HLA-matched related (n=7) or unrelated (n=7) hematopoietic cell grafts from marrow (n = 8), peripheral blood stem cell (n = 5), or umbilical cord blood (n = 1). All patients were given postgrafting immunosuppression with MMF and CSP. Two patients had no pre-transplant conditioning, whereas host immunosuppression was provided by 200 cGy TBI alone (n=3) or in combination with 90 mg/m2 fludarabine (n=9) before hematopoietic cell transplantation (HCT). Mixed (n=5) or full (n=5) donor cell chimerism was established in 10 patients. Four patients required second HCT (n=3) or donor lymphocyte infusion (n=1) due to either T cell graft rejection (n=1), loss of the granulocyte graft (n=1), or low levels of T cell chimerism (n=2). Mortality by days 100 and 180 was 0%. The cumulative incidences of grade II–IV and III–IV acute graft versus host disease (GVHD) were 79% and 14%, respectively. The 1-year incidence of extensive chronic GHVD was 64%. Median follow up for the living recipients was 3 (range, 0.8–6.4) years. The 2 year overall survival, event free survival and transplant related mortality were 68%, 60%, and 16%, respectively. In 7 of the 10 patients with stable donor engraftment correction of immune dysfunction was documented. Specifically, the 4 patients with SCID (n=3) and CVID (n=1) had normal T cell numbers and function following HCT. One patient with WAS had correction of T cell function and platelet numbers after HCT; however subsequently died of B cell non-EBV lymphoma. In addition, 1 patient with CD40LD showed increases in functional CD40 ligand expressing T-cells and response to a neoantigen. Finally, 1 patient with CGD exhibited a normal neutrophil oxidative burst following HCT. Three patients (TCD n=2, CD40LD n=1) were not evaluable for disease response due to continued immunosuppression for chronic GVHD. These results indicate that postgrafting immunosuppression plus nonmyeloablative HCT markedly reduces early HCT related mortality among high-risk patients with immunodeficiency disorders.

Comprehensive Immune Reconstitution without Development of Graft Versus Host Disease (GvHD) Using Limited T-Cell Add Back at the Time of Unrelated Multi-Antigen Mismatched Pan-T-Cell Depleted Transplant.

Application of unrelated multi-antigen mismatched transplant is limited by a high risk of severe GvHD. Pan-T-Cell depletion successfully limits GvHD in this setting, but graft failure, early disease relapse and prolonged immunodeficiency have prevented broad use of this approach. In an attempt to limit GvHD, obtain engraftment and early disease control, we evaluated the effect of adding back 1 x 105 bone marrow derived CD3+ cells/kg patient body weight, following full conditioning, to a pan-T-cell depleted (Isolex, Baxter, Chicago IL) marrow graft (1 x 106 CD34+ cells/kg). In this pilot trial, three consecutive male patients with high risk malignancies without matched donors (disease/age: ALL-CR2/22, refractory AML/31 and CML blast crisis/55 failed imatinib) consented to receive multi-antigen mismatched unrelated marrow grafts (ALL at B/C, AML at B/DR/DQ and CML at A/DQ). Two patients (AML/CML) received transplant conditioning with busulfan/cyclophosphamide and one received TBI/cyclophosphamide (ALL). In addition, all patients received rabbit derived ATG (3 mg/kg days -3 and -2). GvHD prophylaxis included standard tacrolimus and methotrexate with prednisone (0.5 mg/kg starting day +7 tapered off by day + 90). All patients achieved neutrophils 1000/ul (days +15, 21 and 26). No patient developed detectable acute or chronic GvHD. One patient (ALL) died on day +110 in remission, from drug resistant CMV pneumonia. The other two are long term clinical disease-free survivors at 25 (CML) and 40 months (AML) with complete blood counts and differentials within the normal range. Neither survivor has experienced any serious late infection and both have undetectable EBV DNA. Immunoglobulin levels (IgG, A and M) are within the normal range for both patients except for a low IgA level (29 mg/dl) in the CML patient. At 40 months post-transplant, the AML patient is 100% female by FISH, and has normal T and B cell counts (CD3, CD8, helper/suppressor ratio, CD19) with a borderline low CD4 count(507; nl range >666cells/cu.mm). At 25 months, the CML patient has normal CD3 and CD8 counts, increasing CD4 counts (260) and improving chimerism (CD33+ 100% donor; CD3+ 33% donor). TCR Vb CDR3-size spectratype analysis of CD4 and CD8 subsets of these two patients were compared to 6 healthy donors to assess the complexity of their reconstituting T cell repertoire. Of 24 Vb families, 21–23 were resolvable revealing an overall complexity in the CD4 compartment of 91% (AML) and 82% (CML), and in the CD8 compartment 86% (AML) and 73% (CML). Limited T-cell add back at the time of unrelated multi-antigen transplant did not cause GvHD, was associated with prompt and stable engraftment and appears to facilitate comprehensive immune reconstitution. Further study is warranted.

Efficacy and Safety of Using G-CSF on Day before Stem Cell Infusion (Day -1) in Hematopoietic Stem Cell Transplantation (HSCT) -A Single Institution Experience from India.

Background: It is well established that recombinant human G-CSF accelerates neutrophil recovery following hematopoietic stem cell transplantation. However, the optimal timing of G-CSF following transplantation remains unknown. We have done a Phase II non randomized open label study on effects of adding an extra dose of G-CSF (filgrastim 5μgm/kg) on Day -1 of HSCT.Patients and methods: Thirty nine consecutive patients who underwent HSCT in one year period were given G-CSF on Day -1(Day before stem cell infusion) in addition to the regular doses from Day +1 till engraftment.Results: Among the 39, males were 31 and females 8 with 15 allograft and 24 autografts. The type of harvest was PBSC in 35, BM alone in 1and both in 3 cases. Disease wise distribution was AML-11, MM-8, NHL-6, HL-5, CML-4, MDS-2 and Aplastic Anemia, ALL and Ca nasopharynx 1 each. The median MNC and CD 34 doses infused were 5.4X108/Kg and 2.26X106/Kg respectively. Median days of Grade IV neutropenia was 11 (range 5–19) and that of ANC< 100 was 6 (range 2–19). Neutropenic fever was present for a median of 10 days (range 4–19). Neutrophil engraftment and platelet engraftment occurred at Day +11(range 8–13) and Day +21(range 11–73) respectively. Median number of packed cells and platelets (SDP) transfused were 4 and 5 respectively. Mucositis was present for a median of 9 days with Grade III in 51% (n=20) and Grade IV in 23% (n=9) patients. TPN was used in 76% (n=30) patients for a median days of 6 days. Median number of days of antibiotics use was 10 and 57% (n=22) needed 3 lines of antibiotics. Antifungals were used in 57% (n=22) and 95% of use was empirical. Infections were documented in 42% (n=16). Median days of hospitalization was 22 (range 13–38). Transplant related mortality was 10% (2 Auto and 2 Allo). After a median follow up of 113 days 79.71% (n=34) patients are alive with 71% (n=28) in complete remission. One patient with Ca nasopharynx had progressive disease on evaluation and one patient with peripheral T cell lymphoma developed a secondary leukemia (myeloid) post transplant.Conclusions: Addition of G-CSF on Day -1 of HSCT is safe and provides stable engraftment and acceptable results in terms of neutropenic fever, requirement of blood products, antibiotics, TPN and duration of hospital stay. It needs to be compared in randomized trials with use of G-CSF from Day +1 onwards to prove its superiority over that schedule.

AMD3100 + G-CSF Improves Hematopoietic Progenitor Cell (HPC) Collection in Patients with Hodgkin's Disease (HD).

Autologous stem cell transplantation is indicated for patients with HD who have primary refractory disease or who relapse after a first remission. For these patients, as for other patients undergoing autologous transplantation, the number of CD34+ cells infused is a reliable predictor of neutrophil and platelet engraftment, and doses ≥ 5 x 106 CD34+ cells/kg are associated with faster count recovery. However, among the 98 patients with HD who have undergone G-CSF-alone mobilization at our institution in the past five years, 22% did not achieve a minimum HPC collection of 2 x 106 CD34+ cells/kg in ≤ 5 apheresis procedures, and only 15% achieved a collection ≥ 5 x 106 CD34+ cells/kg. AMD3100 mobilizes HPCs by reversibly inhibiting the interaction of CXCR4 and SDF-1α. It has been shown to improve HPC mobilization in patients with multiple myeloma and non-Hodgkin’s lymphoma. Here we present results for the first ten HD patients treated with a mobilization regimen of AMD3100 + G-CSF. To date, ten patients with relapsed (8) or refractory (2) HD have been mobilized with G-CSF (10 ug/kg/d) + AMD3100 (240 ug/kg/d) beginning on day 4. Apheresis was performed 11 hours after each AMD3100 dose. The first dose of AMD3100 produced a median (range) 3.0 (1.9–5.1) fold increase in the number of circulating CD34+ cells. Six patients (60%) achieved a collection of ≥ 5 x 106 CD34+ cells/kg, and all patients collected &gt; 2 x 106 CD34+ cells/kg (range, 3.6–9.4 x 106 CD34+ cells/kg). The median (range) number of apheresis procedures performed per patient was 2 (1–4). No grade II-IV adverse events were ascribed to AMD3100. Eight patients have been transplanted with G-CSF + AMD3100 mobilized cells. All have had prompt and stable engraftment, with median neutrophil recovery at day +9 (9–11) and median platelet recovery at day +20 (15–23). Two patients had very early engraftment, with absolute neutrophil count greater than 100 on day +7. We conclude that AMD3100 + G-CSF is a well-tolerated and effective mobilization regimen in patients with HD. All patients (100%, 95% CI 69%-100%) mobilized with AMD3100 + G-CSF achieved the minimum collection of 2 x 106 CD34+ cells/kg, and a significantly higher proportion of patients (60%, 95% CI 26%–88%) achieved the goal collection of ≥ 5 x 106 CD34+ cells/kg than did the historical controls (15%). Importantly, the median collection in the first two days of pheresis was 5.4 x 106 CD34+ cells/kg, which is significantly better than historical controls, who collected a median 3.0 x 106 CD34+ cells/kg in the first two days of pheresis (p=0.014). Our results demonstrate that the mobilization regimen of AMD3100 + G-CSF can improve the number of HPCs collected and decrease the number of days of pheresis in HD patients. This regimen will be pursued further in this patient population.

Genomic Mapping of Retrovirus Integration Profiles after In Vivo Selection of Chemo-Protected Stem Cells in the Clinically Relevant Canine Model.

In vivo selection strategies that convey a survival advantage to genetically modified cells carrying mutant forms of MGMT (P140K) have the potential to improve autologous and allogeneic stem cell gene therapy and transplantation. Previously, we have shown efficient in vivo selection and marrow protection in a clinically relevant canine model. Here we describe retrovirus integration site (RIS) mapping in the canine genome after chemotherapy with O6BG and BCNU or temozolomide (TMZ). This study includes 4 dogs that received chemotherapy (2 allogeneic and 2 autologous transplants) and an untreated control dog. Animals received CD34-enriched marrow cells transduced with an RD114-pseudotype retroviral vector expressing a bicistronic message containing P140K and GFP while the control dog retroviral vector encoded only GFP. After stable engraftment 4 dogs were treated with O6BG and either BCNU or TMZ. Initial granulocyte marking (10–16%) was increased to >98% with dose-escalating chemotherapy and stabilized at 66–97% with stable increases in all cell lineages analyzed. For LAM-PCR DNA was extracted from peripheral blood leukocytes after various rounds of chemotherapy. The DNA samples analyzed for the control animal corresponded chronologically with that of the chemotherapy treatment animals. Criteria for a ‘true' RIS are a BLAT (http://genome.ucsc.edu/ ) score of 35 or greater, >90% identity to the canine genome (actual identity >99%) and the second ranked score <98% of the first score. To map the RIS relative to genes in the canine genome we utilized alignment of human RefSeq genes to the canine genome (UCSC BLAT: xenoRefFlat). It has been previously described that gammaretrovirus integrate preferentially around promoter and enhancer regions and that is recapitulated in our data. Analysis of a quadrant <5kb around transcription start sites contains 13.3% and 25.5% of the total RIS sites analyzed for the chemotherapy and control animals respectively and when the quadrant is increased to (+/−)50kb that respectively comprises 60.0% and 75.0% of the RIS. We cataloged oncogenes <50kb from RIS to determine if chemotherapy has selected for potentially pre-malignant clones. Interestingly, this does not seem to be the case and in fact the only oncogene identified <50kb from a RIS in a chemotherapy animal (LASP1) was only isolated from a small subset of samples before chemotherapy was administered. In the control animal we identified three oncogenes (TAL1, LYL1 and TFG) within 50kb of a RIS. This suggests that chemotherapy has not selected for clones in or near oncogenes beyond that which already occur for gammaretroviruses. Initial RIS analysis of CFUs has shown that the average copy number is 1.52 and 1.46 for chemotherapy and control animals respectively, suggesting multiple integration events are not required for chemo-protection. We are in the process of carrying out a more thorough analysis of RIS before and after chemotherapy and will track the contribution to hematopoiesis of specific clones using real time-PCR. Our studies suggest that after aggressive in vivo selection of chemo-protected cells multiple clones contribute to hematopoiesis and no side effects or malignancies were observed. These studies and future studies in the nonmyeloablative setting are required to assess the long-term safety of chemo-protective gene therapy.

Stable Mixed Hematopoietic Chimerism in Dogs Conditioned with Donor Antigen, Anti-CD154 Antibody and 100cGy TBI.

Stable mixed chimerism can be established in dogs given a sublethal dose of 200cGy total body irradiation (TBI) before and immunosupression with mycophenolate mofetil (MMF) and Cyclosporine (CSP). When the TBI dose is reduced to 100 cGy, only transient engraftment is observed. Here we asked whether stable engraftment after 100 cGy TBI could be accomplished by first reducing the intensity of host immune responsiveness with the help of an anti-CD154 antibody which blocks the CD40-CD154 pathway.Accordingly, recipients were given a single i.v. injection of 5 mg/kg anti-CD154 antibody (day -5) followed one day later by combined i.v./sc. injections of 107 peripheral white blood cells/kg from their intended DLA-identical littermate marrow donors. TBI, 100cGy (delivered at 7 cGy/min), was given on day 0 followed by infusion of a median of 4.22x108 marrow cells/kg. Postgrafting immunosuppression consisted of MMF (10mg/kg, BID, days 0 to 28) and CSP (15 mg/kg BID, days -1 to 35). Six dogs have been transplanted. Two dogs are too early after transplant for evaluation, while four dogs have been followed for >9, 12, >18 and >26 weeks. All four dogs had initial engraftment. Three of four dogs have continued to show stable mixed donor/host hematopoietic chimerism among lymphocytes and granulocytes from the peripheral blood. One dog rejected the graft 12 weeks after transplantation. All six transplanted dogs are alive and well. Besides mild allergic reactions to the antibody in five of the six dogs (redness of the eyes, skin rashes and dry mucous membranes), no additional side effects attributed to the pretransplant treatment were observed.Data were consistent with previous findings in dogs, in which the costimulatory signal between B7 and CD28 was blocked, and support the hypothesis, that stable marrow allografts would be established by combining non-myeloablative pretransplant host immunosuppression and posttransplant immunosupression of both host and donor cells using MMF and CSP.

Daily Monitoring of Cyclosporine (Csa) Allows Better Control of Graft vs. Leukaemia Effect (GvL): Single Centre Experience.

The most important consideration in modern SCT practices is the fine balance that is required to achieve adequate immunosuppression and stable engraftment thus allowing the platform to mount an effective GVL response. Though CsA facilitates this objective, it is associated with several adverse clinical manifestations due to its narrow therapeutic index (TI) and wide variability in the absorption kinetics. Weekly monitoring of trough CsA levels are used to adjust the dose. The aim of this study is to determine whether daily monitoring of CsA levels enables better achievement of these goals and yet reduce the serious toxicities.Patients & methods: Twenty three patients (M=15, F=8, median age 46yrs range 19–63) received an allogeneic transplant (sibling-15, MUD-7 mismatched unrelated-1) since March 2004 when we started to monitor daily CsA levels (Biostat TDX analyser using fluorescent immunoassay) from the day of first administration until the time of discharge. The transplants included full intensity (FI=11), reduced intensity (RI=11) or customised (1) for various haematolymphoid malignancies. Patients received uniform GVHD prophylaxis with IV CsA (RI+FI) and methotrexate (FI) as per standard loading doses. The CsA target level was adjusted to 200–220 ng/ml from Day-1 onwards and was later modified according to the treatment strategy. Oral CsA administration was started when patients were able to switch over. Results Nine of 23 (40%) patients developed acute GVH (n=7 grade1–2, n=2 grade 3–4) at a median of 23.5 days (10–28). Two of 9 patients required addition of steroids by Day+30. The median CsA level at the first occurrence of GVH was 247ng/ml (113–333) and the median CsA level during hospitalisation was 207 ng/ml (136–291). Only one patient who received T cell addback required increased dose of IV CsA to manage Grade 4 GVH. Twenty two/22 patients engrafted with a median day to neutrophil recovery being 13 days (8–35). One patient lost his graft due to a low initial cell dose and the inability of the MUD donor to donate any further cells. There was no significant difference in the baseline and Day30 creatinine levels (median highest creatinine 108μmol/l range 58–267) or bilirubin levels (median highest bilirubin 27μmol/l, range15–251). Five/22 patients developed CMV reactivation/infection and 1 developed RSV infection. No patients developed seizures and in one patient pre-existent neuropathy was exacerbated which was directly attributable to CsA. Twenty one/22 patients (95.5%) switched to oral CsA by Day+25. In 8/22 (36.36%) patients, the target CsA levels was around 100–150 ng/ml at the time of discharge. The median duration of hospital stay was 35days (24–130).Conclusions: These observations suggests that daily monitoring of CsA is safe and successful. This allowed successful engraftment and a more liberal management of Grade 1–2 GVH thus allowing us to maximise the GVL effect. Despite concomitant use of other toxic drugs, side effects were minimal and reversible. Close observation of early clinical manifestations enabled us to modify the immunosuppressive strategy at later time periods. This approach also allowed us to better manage patients suffering from CMV and EBV/PTLD (patient number 10 on day+88) by reducing the dose during active infections.

Improved Analysis of Hematopoietic Engraftment by Non-Invasive In Vivo Bioluminescent Imaging of Transplanted Human Embryonic Stem Cell-Derived Hematopoietic Cells.

Animal transplantation models are essential to characterize the long-term in vivo engraftment capacity of putative hematopoietic stem cells derived from human embryonic stem cells (hESCs). We have previously demonstrated that hESCs can be routinely utilized to derive multiple hematopoietic cell lineages. Here, we use in vivo bioluminescence imaging (BLI) of stable luciferase (luc)-expressing hESCs to noninvasively monitor the dynamics of transplantation, engraftment, and growth of hESC-derived hematopoietic cells within individual animals over an extended time course. Luc expression under control of an EF1α promoter was introduced into the H1 hESC line using a self-inactivating lentiviral vector. Undifferentiated hESC colonies that stably expressed luciferase were established and selected, and the pluripotent capability of luc+ hESCs was first explored by teratoma formation. Undifferentiated luc+ human ES cells were intramuscularly injected into NOD/SCID mice. The dynamics of survival and growth of the hESCs was monitored by BLI using the IVIS Imaging System (Xenogen) at regular time points post-transplantation. There was a decrease of luminescent signal during the first 1–2 weeks. This was followed by a dramatic increase in luminescent signals after about 5 weeks, which correlated with teratoma size. Immunohistochemical analysis confirmed stable luc-expression in multiple differentiated cell types within the teratomas. We next used BLI to examine luc+ H1 hESCs that were induced to undergo hematopoietic differentiation by co-culture with S17 cells, to give rise to H1/S17 cells. Flow cytometric studies confirmed hematopoietic cells (CD34+, CD45+, CD31+, and c-kit+ cells) were derived from these differentiated luc+ hESCs, with 5–10% of H1/S17 cells being CD34+. Hematopoietic progenitors that gave rise to colonies of mature luc+ blood cells in a standard CFU assay were also observed from the H1/S17 cells. Luc-expression of differentiated hESCs was maintained at similar levels to those of the undifferentiated ES cells. To define the in vivo potential of luc+ hESC-derived hematopoietic cells, hESCs were allowed to differentiate on S17 cells for two weeks. SCID-repopulating cell studies were done by intravenous (iv) injection into sublethally irradiated NOD/SCID mice. After iv injection of 2–3 x106 unsorted luc+ H1/S17 cells, BLI showed the brightest signal in the lung at day 0 (within 2 hours), followed by a rapid decline in signal on the next day (day1). On day 8, most luc+ cells were detected in the abdomen and liver. Subsequently, after 6–12 weeks, multiple engraftment loci were identified in hematopoietic tissues. Flow cytometric analysis of bone marrow from these mice confirmed the presence of hESC-derived human CD45+ cells. Engraftment was also demonstrated after direct intra-bone marrow injection with as few as 60,000 CD34+ cells sorted from luc+ H1/S17 cells. Again, stable engraftment can be monitored by BLI for 8+ weeks. These results demonstrate that BLI has several important advantages as an effective non-invasive approach to track and quantitatively monitor in vivo engraftment of hematopoietic or other cell lineages derived from hESCs.

DLA-Haploidentical Stem Cell Allografts after Anti-CD44 Therapy and Nonmyeloablative Conditioning: The Impact of Donor Lymphocyte Infusion (DLI), Pentostatin and Graft Composition on Donor Chimerism and Rejection.

Background: We previously reported that engraftment across a dog leukocyte antigen (DLA) haplotype-mismatched barrier could be achieved after nonmyeloablative conditioning with 200 cGy total body irradiation (TBI) plus post-grafting mycophenolate mofetil (MMF) /cyclosporine (CSP) when anti-CD44 monoclonal antibody (MAb), S5, is added to the regimen. However, 50% of dogs rejected their grafts after discontinuation of MMF/CSP. Here, we studied the influence of donor lymphocyte infusion (DLI), pentostatin, and graft composition in achievement of sustained full donor chimerism and prevention of rejection.Methods: Twenty-nine dogs were administered MAb S5 (0.2 mg/kg/day) from days -7 to -2, before 200 cGy TBI. Unmodified G-CSF-mobilized peripheral blood stem cells (PBSC) from DLA-haploidentical donors were infused followed by immunosuppression with MMF (5-10 mg/kg BID SQ) and CSP (15 mg/kg BID PO). Dogs in group 1 (n=8) were not given DLI and dogs in group 2 (n=5) were given 6 doses of pentostatin (4 mg/m2/dose) before TBI and no DLI. Dogs in group 3 (n=10) and 4 (n=6) received escalating doses of DLI on days +35, +63, +92. In addition, dogs in group 4 received pentostatin (4 mg/m2) 2 days prior to each infusion of DLI.Results: All dogs achieved initial engraftment between 1 to 2 weeks after transplant and survived 5 weeks or longer with mononuclear cell chimerism ranging from 2% to 98% (median 37%) on day +35. Higher cell subset levels in the PBSC graft were associated with stable donor engraftment: total nucleated cells (p=0.03), CD4+ (p=0.01), CD8+ (p=0.03) and CD14+ (p=0.009) cells. Also, there was a trend in the number of CD34+ (p=0.10) and CD3+(p=0.07) cells. Graft rejection was seen in 7 of 13 dogs without DLI (groups 1 and 2) and in 7 of 16 dogs that received DLI (groups 3 and 4). There was no statistically significant association between DLI and rejection (p=0.44). Six of 16 dogs who received DLI and 1 of 13 dogs with no DLI achieved full donor chimerism (p=0.18), yet there was no statistically significant association between the four groups (p=0.52). There was a trend for higher counts of CD4+ and CD8+ cells (p=0.08) and achievement of full donor chimerism. However, the use of pentostatin before each DLI (group 4) did not impact achievement of full donor chimerism (p>0.95). Duration of mixed chimerism was associated with higher cell subset levels of infused PBSC; CD34+ (p=0.06), CD4+ (p=0.02), CD14+ (p=0.05), CD3+ (p=0.09), CD8+ (p=0.08).Conclusions: The use of DLI with or without prior infusion of pentostatin after initial engraftment of DLA-haploidentical donor graft did not facilitate conversion to full donor chimerism, sustain mixed chimerism, or prevent graft rejection. However, the infusion of haploidentical donor PBSC graft with higher numbers of TNC, CD34+, CD3+, CD4+, CD8+ and CD14+ cells could prolong donor chimerism and prevent graft rejection.

Boosts with Highly Purified Stem Cells Can Improve Hematopoietic Regeneration after Allogeneic Transplantation from Alternative Donors.

In allogeneic transplantation it is a major goal to maintain a stable long term engraftment. However, several factors like viral infections, drug toxicity or low stem cell numbers may hamper hematopoietic recovery or may even cause a late graft failure after successful initial engraftment. Hematopoietic growth factors or additional stem cell donations (boosts) may help to overcome this problem. In this retrospective analysis, the efficacy of boosts with highly purified peripheral stem cells either from closely matched unrelated or from haploidentical related donors was evaluated in pediatric patients with stagnant hematopoiesis (<1500 leucocytes or <50 000 platelets between 30–90 days posttransplant, n=11) or with initially increasing and then decreasing hematopoiesis (n=3). Patients with Graft rejection or hemophagocytosis were excluded. Diagnoses were: acute leukemias (n=10), nonmalignatn diseases (n=4). Most boosts were taken from the original graft: if the grafts contained >10x106 (unrelated donors) or >20x106/kg BW CD34+ cells, surplus stem cells were purified with an immunomagnetic selection method using antiCD34 or antiCD133 coated microbeads and cryopreserved. Three haploidentical donors were asked for a second donation. Our aim was to maintain stable and complete hematopoiesis after transplantation without inducing acute or chronic GvHD.A total of 18 boosts were given between 30 and 290 days after transplantation with a median number of 5 (0.6–34) x 106 CD34+/CD133+ progenitor cells/kg and only 2200 (100–25000) residual T-cells/kg. All patients had a complete donor chimerism and no reconditioning or posttransplant pharmacological immunosuppression was administered. Patients who received G-CSF were excluded. Due to the low number of T cells, acute GvHD >grade I and also chronic GvHD could be completely avoided even in haploidentical donors.Leukocyte counts, neutrophile counts, lymphocyte counts (and platelet counts in patients with thrombocytopenia) within the week before infusion of the boosts were compared with cell counts 4 and 8 weeks after infusion, using the paired T-test. A significant increase of all cell counts was observed (medina numbers pre boosting: 900/μl, 312/μl, 142/μl; 4 weeks post boosting: 2065/μl, 1264/μl, 317/μl; 8 weeks post boosting:2570/μl, 1780/μl, 385/μl; p<0.05 for all comparisons). Furthermore, independence from platelet transfusion could be achieved.Conclusions: Boosts with purified stem cells and low T-cell contamination (<10000 T-cells/kg) can be administered after transplantation without the risk of inducing GvHD, even in mismatched donors. No pharmacological immunosuppression was necessary. Thus, the method represents a safe tool and can improve donor derived hematopoiesis.