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Preclinical and early phase clinical studies suggest that an appropriately dosed umbilical cord blood (CB) infusion has the potential to help improve motor function in young children with cerebral palsy (CP). As many children with CP do not have their own CB available, use of allogeneic cells would extend access to this potentially beneficial therapy to more children. In this phase I, open-label study, 15 children, aged 1 to 6 years, with moderate to severe spastic CP were treated with a single intravenous infusion of allogeneic human leukocyte antigen (HLA) matched or partially matched sibling CB with a cell dose of ≥2.5 × 107 cells/kg based on the pre-cryopreservation count (median infused cell dose, 3.3 × 107; range, 1.8-5.2 × 107). There were a total of 49 adverse events (AEs) over a 2-year time period, but there were no AEs related to the CB infusions. Specifically, there were no acute infusion reactions and no antibody formation against platelets, red blood cells, or donor-specific HLA antigens. Donor cells were not detected in peripheral blood 6 months later. Six months after infusion, participants were assessed for response and experienced a mean ± SD increase of 4.7 ± 2.5 points on the Gross Motor Function Measure-66 and 1 ± 2.9 points on the Peabody Gross Motor Quotient. Appropriately dosed, allogeneic partially or fully HLA-matched sibling CB infusion is well tolerated and potentially beneficial in young children with CP.

Prophylactic donor lymphocyte infusions (DLI) are used to augment post-transplant immune recovery to reduce both infectious complications and disease recurrence. Preclinical studies implicate the naive T-cell subset as the primary driver of graft-versus-host disease (GvHD). In this phase I dose escalation study, we assessed the safety of a DLI that was depleted of CD45RA+ naive T cells. Sixteen adult patients received a prophylactic DLI at a median of 113 days (range 76–280 days) following an HLA-identical, non-myeloablative allogeneic hematopoietic stem cell transplantation. Three patients each received the naive T-cell depleted DLI with a CD3+ dose of 1 × 105/kg, 1 × 106/kg, and 5 × 106/kg. The maximum dose of 1 × 107/kg was expanded to 7 patients. No dose-limiting grade III/IV acute GvHD or adverse events attributable to the DLI were observed at any dose level. One patient developed grade 2 acute GvHD of skin and upper intestines, and another developed moderate chronic GvHD of the lungs following the DLI. With a median follow-up of 2.8 years, 2-year progression-free and overall survival is 50.0% and 68.8%, respectively. In conclusion, these data suggest that a DLI that has been depleted of CD45RA+ naive T cells is feasible and carries a low risk of acute or chronic GvHD.

BACKGROUND The cell dose infused for cord blood transplantation strongly correlates with outcomes following transplantation. Post thaw recoveries can be improved by washing cord blood units (CBUs) in dextran/albumin. Early methods used a labor-intensive manual process. We have recently developed and validated an automated washing method. We now report our results of a study comparing cellular recoveries achieved after manual and automated wash, as well as the impact on engraftment following allogeneic transplantation. STUDY DESIGN AND METHODS CBUs distributed by the Carolinas Cord Blood Bank for clinical use at Duke University after manual or automated wash were included in this report. Precryopreservation total nucleated cell count, total CD34+, colony-forming units, recoveries, and sterility were analyzed by wash method. Patient age, cell dose/weight, diagnosis, conditioning regimen, immunosuppression, and time to neutrophil engraftment were also analyzed. RESULTS Manual and automated washed CBUs yielded similar total nucleated cell count and total CD34+ recoveries. Significantly higher colony-forming units recoveries were achieved after automated washing. Patients who received CBUs washed via an automated method experienced earlier neutrophil engraftment. CONCLUSION While manual and automated washing achieved similar post thaw cellular recoveries, automated washed CBUs demonstrated higher colony-forming unit recovery, which is an important predictor of potency and engraftment. Furthermore, we demonstrated that automated washing was associated with earlier neutrophil engraftment. Our findings favor the use of an automated wash method over a manual approach.

Cerebral palsy (CP) is a condition affecting young children that causes lifelong disabilities. Umbilical cord blood cells improve motor function in experimental systems via paracrine signaling. After demonstrating safety, we conducted a phase II trial of autologous cord blood (ACB) infusion in children with CP to test whether ACB could improve function (ClinicalTrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT01147653","term_id":"NCT01147653"}}NCT01147653; IND 14360). In this double‐blind, placebo‐controlled, crossover study of a single intravenous infusion of 1–5 × 107 total nucleated cells per kilogram of ACB, children ages 1 to 6 years with CP were randomly assigned to receive ACB or placebo at baseline, followed by the alternate infusion 1 year later. Motor function and magnetic resonance imaging brain connectivity studies were performed at baseline, 1, and 2 years post‐treatment. The primary endpoint was change in motor function 1 year after baseline infusion. Additional analyses were performed at 2 years. Sixty‐three children (median age 2.1 years) were randomized to treatment (n = 32) or placebo (n = 31) at baseline. Although there was no difference in mean change in Gross Motor Function Measure‐66 (GMFM‐66) scores at 1 year between placebo and treated groups, a dosing effect was identified. In an analysis 1 year post‐ACB treatment, those who received doses ≥2 × 107/kg demonstrated significantly greater increases in GMFM‐66 scores above those predicted by age and severity, as well as in Peabody Developmental Motor Scales‐2 Gross Motor Quotient scores and normalized brain connectivity. Results of this study suggest that appropriately dosed ACB infusion improves brain connectivity and gross motor function in young children with CP. Stem Cells Translational Medicine 2017;6:2071–2078

Prophylactic donor lymphocyte infusions (DLI) are used to augment post-transplant immune recovery to reduce both infectious complications and disease recurrence. Preclinical studies implicate the naive T-cell subset as the primary driver of graft-versus-host disease (GvHD). In this phase I dose escalation study, we assessed the safety of a DLI that was depleted of CD45RA+ naive T cells. Sixteen adult patients received a prophylactic DLI at a median of 113 days (range 76-280 days) following an HLA-identical, non-myeloablative allogeneic hematopoietic stem cell transplantation. Three patients each received the naive T-cell depleted DLI with a CD3+ dose of 1 × 10

Ongoing neuroinflammation may contribute to symptoms of autism spectrum disorder (ASD) in at least a portion of affected individuals. Mesenchymal stromal cells (MSCs) have demonstrated the capacity to modulate neuroinflammation, but safety and feasibility of MSC administration in children with ASD have not been well established. In this open‐label, phase I study, 12 children with ASD between 4 and 9 years of age were treated with intravenous (IV) infusions of human cord tissue mesenchymal stromal cells (hCT‐MSCs), a third‐party MSC product manufactured from unrelated donor umbilical cord tissue. Children received one, two, or three doses of 2 × 106 cells per kilogram at 2‐month intervals. Clinical and laboratory evaluations were performed in person at baseline and 6 months and remotely at 12 months after the final infusion. Aside from agitation during the IV placement and infusion in some participants, hCT‐MSCs were well tolerated. Five participants developed new class I anti‐human leukocyte antigen (HLA) antibodies, associated with a specific lot of hCT‐MSCs or with a partial HLA match between donor and recipient. These antibodies were clinically silent and not associated with any clinical manifestations to date. Six of 12 participants demonstrated improvement in at least two ASD‐specific measures. Manufacturing and administration of hCT‐MSCs appear to be safe and feasible in young children with ASD. Efficacy will be evaluated in a subsequent phase II randomized, placebo‐controlled clinical trial., In this phase I study, intravenous infusion of human cord tissue mesenchymal stromal cell was safe and feasible in young children with autism. Autism symptoms improved in half of children, and 40% developed clinically silent but detectable anti‐human leukocyte antigen antibodies post‐infusion.

Introduction An expanded access protocol (EAP) of autologous or sibling umbilical cord blood (CB) infusion was established in 2017 under Investigational New Drug #15949 to provide access to this investigational procedure for individuals with certain neurological conditions who do not qualify for available clinical trials. Objective The objective was to report updated enrollment and safety data from a single center EAP of autologous/sibling CB infusion for children with acquired neurological conditions. Methods Children with autism, cerebral palsy, and other related conditions were screened remotely under a separate institutional review board-approved screening protocol, including review of parental questionnaires, medical records, basic labs, and CB unit reports. Based on emerging clinical trial data, the minimum cell dose was increased to a total nucleated cell count (TNCC) >2.0 × 107/kg. CB units also had to have viability >70%, negative sterility cultures and maternal donor screening labs, and a segment available for identity confirmation and potency testing, and sibling units had to be at least human leukocyte antigen-haploidentical. CB units were thawed, washed, and infused via peripheral IV after premedication with IV diphenhydramine and methylprednisolone. Some children received repeated doses, when the TNCC allowed. Results A total of 2,001 children were screened from November 2017 to June 2021, and 464 children received 494 CB infusions under the EAP, including 20 children who had previously participated in a clinical trial and elected to return to receive a subsequent infusion. Due to COVID restrictions, enrollment was suspended for 18 weeks (March 11, 2020, to July 21, 2020). Patient and CB characteristics are shown in Table 1. Infusions were generally well-tolerated. Forty-one (9%) adverse events, all hypersensitivity reactions, were probably or definitely related to CB infusion. Serious adverse events were documented in 8 children, all expected and unrelated to CB infusion. There were 6 (0.01%) positive post-thaw cultures;none developed infections or required antibiotics. Parental surveys were completed 1 year (n = 244, 72%) and 2 years (n = 105, 51%) post-infusion. Parental assessment varied regarding clinical improvement. Discussion Autologous/sibling CB infusions conducted under an EAP for children with neurological conditions are safe and feasible with varied reported responses to the infusions. CB eligibility was amended to incorporate early phase clinical trial outcomes regarding cell dose. Efficacy continues to be evaluated in ongoing clinical trials.

Objective To evaluate whether umbilical cord blood (CB) infusion is safe and associated with improved social and communication abilities in children with autism spectrum disorder (ASD). Study design This prospective, randomized, placebo-controlled, double-blind study included 180 children with ASD, aged 2-7 years, who received a single intravenous autologous (n = 56) or allogeneic (n = 63) CB infusion vs placebo (n = 61) and were evaluated at 6 months postinfusion. Results CB infusion was safe and well tolerated. Analysis of the entire sample showed no evidence that CB was associated with improvements in the primary outcome, social communication (Vineland Adaptive Behavior Scales-3 [VABS-3] Socialization Domain), or the secondary outcomes, autism symptoms (Pervasive Developmental Disorder Behavior Inventory) and vocabulary (Expressive One-Word Picture Vocabulary Test). There was also no overall evidence of differential effects by type of CB infused. In a subanalysis of children without intellectual disability (ID), allogeneic, but not autologous, CB was associated with improvement in a larger percentage of children on the clinician-rated Clinical Global Impression-Improvement scale, but the OR for improvement was not significant. Children without ID treated with CB showed significant improvements in communication skills (VABS-3 Communication Domain), and exploratory measures including attention to toys and sustained attention (eye-tracking) and increased alpha and beta electroencephalographic power. Conclusions Overall, a single infusion of CB was not associated with improved socialization skills or reduced autism symptoms. More research is warranted to determine whether CB infusion is an effective treatment for some children with ASD.

Background & Aim The Marcus Center for Cellular Cures (MC3) at Duke University recently completed a Phase I clinical trial (NCT03099239) using human cord tissue-derived mesenchymal stromal cells (hCT-MSCs) for the treatment of autism spectrum disorder. For this trial, 12 patients ages 2 – 11 years were sequentially dosed with 1, 2, or 3 doses of 2 million MSC/kg post thaw administered intravenously every 2 months. A manufacturing process was developed for our GMP facility to produce the required doses in a flexible and reproducible manner. Methods, Results & Conclusion For the trial, 3 lots of hCT-MSCs were manufactured from cord tissue obtained from 3 term healthy male babies delivered after elective routine C-section. After transporting the cord to the Robertson GMP Laboratory, the tissue was cut and digested in a tissue dissociator. The resulting cell suspension was plated in tissue culture flasks for P0 culture in Prime-XV MSC Expansion XSFM culture medium (Irvine Scientific) supplemented with 1% platelet lysate (PL). After harvest, P0 cells were cryopreserved, with a subset of cells expanded for P1 in XSFM without PL, and with a larger cryopreservation and expansion process for P2. For both P1 and P2, cells were seeded at 1000/cm2. Cells at the end of P2 were frozen in multi-compartment cryo-bags at the appropriate concentration for final dose administration. Prior to release to the patient, each lot of P2 hCT-MSC was characterized by flow cytometry for typical MSC markers, tri-lineage differentiation, and the ability to suppress T-cell proliferation. Manufacturing process data for each donor are presented in Table 1. For 2 donors, additional P2 cells were produced from P1 vials during the course of the trial (e.g., “remanufactured” lots) when the original batch was not enough to meet demand. We calculated that after P0 the cells underwent 10.5 doublings on average after the end of P2, with a mean doubling time in P1 – P2 of 31.2 hours. The final thawed and prepared P2 cell product had a mean viability of 75% prior to administration to the patient. Preparations are underway for a Phase II clinical trial and the subsequent need for a larger scale and more efficient manufacturing process. We are developing a streamlined process using closed systems for cell expansion, concentration, and cryopreservation of the MSC product that is semi-automated and will minimize the use of clean room facilities. Comparability of the MSC product using the new process will be confirmed prior to use in the Phase II trial.

Introduction Prophylactic, donor lymphocyte infusion (DLI) is used to augment immune recovery and graft vs. tumor effect following a T-cell depleted alloSCT. However, it carries the risk of inducing severe GvHD. Our pre-clinical murine studies have implicated the naive T-cell, defined by CD62L marker, as the primary driver of alloreactivity. We hypothesize that transferring selected memory T-cells without naive-T cells would endow the patient with cells that do not cause GvHD while augmenting host defense against infection and tumor recurrence. Objectives The goal of the study was to determine the maximum tolerated dose of a prophylactic, delayed DLI that has been depleted of naive T-cells. Methods We enrolled 16 adult patients, median age 54 who met following criteria; a. underwent an alemtuzumab or thymoglobulin-containing non-myeloablative allogeneic transplant from an HLA-identical family donor or an 8/8 HLA-matched unrelated donor (MUD), b. at least 60 days from day of transplantation, c. no active acute GvHD grade II or higher. Primary diseases were CLL; n=1, MM; n=2, MF; n=1, NHL; n=5, MDS; n=1, AML; n=6. A dedicated, non-mobilized donor apheresis procedure was performed following enrollment. Naive, CD45RA+ T-cells were depleted from the collection under an IND using the Miltenyi clinimacs system. A standard phase I, 3+3 dose escalation schema was employed. The dose-limiting toxicity of the DLI was defined as development of grade III/IV acute GvHD within 90 days of the DLI. Results The DLI was infused on median 112.5 days (76 to 280 days) following transplant. 8 patients received matched sibling grafts and 8 received MUD grafts. 3 patients each received naive T-cell depleted CD3+ dose of 1 × 105/kg, 1 × 106/kg, and 5 × 106/kg. The top dose of 1 × 107/kg was expanded to 7 patients. No dose limiting toxicity or adverse event attributable to the DLI was observed at any dose level. 1 patient developed grade 2 acute GvHD and 1 developed moderate chronic GvHD attributable to the DLI. 4 patients had relapsed disease; 2 in the lowest dose and 2 in the highest dose levels. With a median follow-up of 2.75 years, 2-year relapse free and overall survivals are 56.25% and 68.75%, respectively (Fig 1 and 2). NK cells, CD3+T lymphocytes, CD4+ T lymphocytes and CD8+ T lymphocytes show positive correlations of 0.24, 0.40, 0.42 and 0.38 respectively with time following the DLI (Fig 3). Conclusion A prophylactic, naive T-cell depleted DLI can be provided safely, without significant risk of acute GvHD at a dose of 1 × 10e7 CD3/kg. Larger studies in a more homogeneous patient population will be needed to confirm the impact on immune recovery and relapse prevention.

Background aims Removing DMSO post-thaw results in: reduced infusion reactions, improved recovery and stability of viable CD34+ cells. Validated methods use 5%–8.3% Dextran 40 with 2.5%–4.2% HSA for this purpose. Recent shortages of clinical grade Dextran require identification of suitable alternatives. Methods PBPC were used to compare a standard 2X wash medium of 5 parts 10% Dextran 40 in saline (DEX) with 1 part 25% HSA (8.3% DEX/ 4.2% HSA) with Hydroxyethyl Starch (HES)-based solutions. Cells in replicate bags were diluted with an equal volume of wash solution, equilibrated 5 minutes, the bag filled with wash medium, pelleted and the supernatant expressed. Bags were restored to the frozen volume in wash medium and tested by single platform flow cytometry and CFU. Total viability, viable TNC, MNC, and CD34+ cell recovery, and CD34+ cell viability were compared immediately post-thaw and after 90 minutes. Results 5.2% HES/4.2% HSA did not differ from our standard in CD34 recovery or viability. Due to concerns that high concentrations of HES could affect renal function we tested 0.6% HES/2.5% HSA resulting in significantly poorer CD34 recovery and viability. Results improved using 2.4% HES/4.2% HSA and when 0.6% HES/4.2%HSA was used no significant differences were seen. CFU assays confirmed no differences between the standard dextran arm and HES at 2.4% or 0.6% so long as HSA was at 4.2%. Conclusions We conclude that HES from 0.6% to 5.2% with 4.2% HSA is a suitable substitute for Dextran 40 as a reconstitution/washing medium for PBPC products.

BACKGROUND A pilot study was conducted to determine the safety and feasibility of intravenous administration of autologous umbilical cord blood (CB) in young children with acquired neurologic disorders. Most CB units (CBUs) were electively stored in private CB banks. Unlike public banks, which utilize specific criteria and thresholds for banking, private banks generally store all collected CBUs.

Allogeneic stem cell transplantation with umbilical cord blood (UCB) cells is limited by the cell dose a single unit provides recipients. Ex vivo expansion is one strategy to increase the number of cells available for transplantation. Aastrom Biosciences developed an automated continuous perfusion culture device for expansion of hematopoietic stem cells (HSCs). Cells are expanded in media supplemented with fetal bovine serum, horse serum, PIXY321, flt-3 ligand, and erythropoietin. We performed a phase 1 trial augmenting conventional UCB transplants with ex vivo–expanded cells. The 28 patients were enrolled on the trial between October 8, 1997 and September 30, 1998. UCB cells were expanded in the device, then administered as a boost to the conventional graft on posttransplantation day 12. While expansion of total cells and colony-forming units (CFUs) occurred in all cases, the magnitude of expansion varied considerably. The median fold increase was 2.4 (range, 1.0-8.5) in nucleated cells, 82 (range, 4.6-266.4) in CFU granulocyte-macrophages, and 0.5 (range, 0.09-2.45) in CD34+ lineage negative (lin–) cells. CD3+ cells did not expand under these conditions. Clinical-scale ex vivo expansion of UCB is feasible, and the administration of ex vivo–expanded cells is well tolerated. Augmentation of UCB transplants with ex vivo–expanded cells did not alter the time to myeloid, erythroid, or platelet engraftment in 21 evaluable patients. Recipients of ex vivo–expanded cells continue to have durable engraftment with a median follow-up of 47 months (range, 41-51 months). A randomized phase 2 study will determine whether augmenting UCB transplants with ex vivo–expanded UCB cells is beneficial.

We have performed a phase I dose escalation of 4-Hydroperoxycyclophosphamide (4HC) purging of autologous peripheral blood progenitor cells (PBPCs) to improve the outcome of autologous transplantation for patients with myeloid leukemia. Peripheral blood stem cells were mobilized after cytosine arabinoside of 2 g/m2 every 12 hours × 8 doses with etoposide of 40 mg/kg total dose infused over 4 days, followed by growth factor support. The preparative regimen included Busulfan of 1 mg/kg orally every 6 hours × 16 doses, followed by etoposide of 60 mg/kg × 1 day (the patient with chronic myeloid leukemia received cyclophosphamide of 60 mg/kg/d × 2 days in lieu of etoposide). PBPCs purged with 4HC were infused following this induction. Toxicities included grade 3 or 4 skin rashes, stomatitis/mucositis, and delay in time to hematopoietic recovery. The maximum tolerated dose of 4HC used to purge PBPCs in this trial was 20 μg/mL, which resulted in an average of 18 days for white blood cells and 28 days for platelet recovery. With a median follow-up of 2.25 years in surviving patients, the 3-year disease free survival rate is 44% and the overall survival rate is 89%. These data suggest that autologous PBPCs are more sensitive than marrow purged with 4HC, tolerating less intense purging, although a survival advantage may still be seen and should be assessed in larger studies. Approaches to minimize stomatitis and protect normal stem cells from the toxicity of 4HC may improve the tolerance and efficacy of this approach. © 2003 American Society for Blood and Marrow Transplantation Biology of Blood and Marrow Transplantation 9:183-188 (2003)

We have performed a phase I dose escalation of 4-Hydroperoxycyclophosphamide (4HC) purging of autologous peripheral blood progenitor cells (PBPCs) to improve the outcome of autologous transplantation for patients with myeloid leukemia. Peripheral blood stem cells were mobilized after cytosine arabinoside of 2 g/m(2) every 12 hours x 8 doses with etoposide of 40 mg/kg total dose infused over 4 days, followed by growth factor support. The preparative regimen included Busulfan of 1 mg/kg orally every 6 hours x 16 doses, followed by etoposide of 60 mg/kg x 1 day (the patient with chronic myeloid leukemia received cyclophosphamide of 60 mg/kg/d x 2 days in lieu of etoposide). PBPCs purged with 4HC were infused following this induction. Toxicities included grade 3 or 4 skin rashes, stomatitis/mucositis, and delay in time to hematopoietic recovery. The maximum tolerated dose of 4HC used to purge PBPCs in this trial was 20 microg/mL, which resulted in an average of 18 days for white blood cells and 28 days for platelet recovery. With a median follow-up of 2.25 years in surviving patients, the 3-year disease free survival rate is 44% and the overall survival rate is 89%. These data suggest that autologous PBPCs are more sensitive than marrow purged with 4HC, tolerating less intense purging, although a survival advantage may still be seen and should be assessed in larger studies. Approaches to minimize stomatitis and protect normal stem cells from the toxicity of 4HC may improve the tolerance and efficacy of this approach.

BACKGROUND. Delayed hematopoietic recovery is a major drawback of umbilical cord blood (UCB) transplantation. Transplantation of ex vivo–expanded UCB shortens time to hematopoietic recovery, but long-term, robust engraftment by the expanded unit has yet to be demonstrated. We tested the hypothesis that a UCB-derived cell product consisting of stem cells expanded for 21 days in the presence of nicotinamide and a noncultured T cell fraction (NiCord) can accelerate hematopoietic recovery and provide long-term engraftment. METHODS. In a phase I trial, 11 adults with hematologic malignancies received myeloablative bone marrow conditioning followed by transplantation with NiCord and a second unmanipulated UCB unit. Safety, hematopoietic recovery, and donor engraftment were assessed and compared with historical controls. RESULTS. No adverse events were attributable to the infusion of NiCord. Complete or partial neutrophil and T cell engraftment derived from NiCord was observed in 8 patients, and NiCord engraftment remained stable in all patients, with a median follow-up of 21 months. Two patients achieved long-term engraftment with the unmanipulated unit. Patients transplanted with NiCord achieved earlier median neutrophil recovery (13 vs. 25 days, P < 0.001) compared with that seen in historical controls. The 1-year overall and progression-free survival rates were 82% and 73%, respectively. CONCLUSION. UCB-derived hematopoietic stem and progenitor cells expanded in the presence of nicotinamide and transplanted with a T cell–containing fraction contain both short-term and long-term repopulating cells. The results justify further study of NiCord transplantation as a single UCB graft. If long-term safety is confirmed, NiCord has the potential to broaden accessibility and reduce the toxicity of UCB transplantation. TRIAL REGISTRATION. Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01221857","term_id":"NCT01221857"}}NCT01221857. FUNDING. Gamida Cell Ltd.

Objective To assess feasibility and safety of providing autologous umbilical cord blood (UCB) cells to neonates with hypoxic-ischemic encephalopathy (HIE). Study design We enrolled infants in the intensive care nursery who were cooled for HIE and had available UCB in an open-label study of non-cyropreserved autologous volume- and red blood cell-reduced UCB cells (up to 4 doses adjusted for volume and red blood cell content, 1-5 × 107 cells/dose). We recorded UCB collection and cell infusion characteristics, and pre- and post-infusion vital signs. As exploratory analyses, we compared cell recipients' hospital outcomes (mortality, oral feeds at discharge) and 1-year survival with Bayley Scales of Infant and Toddler Development, 3rd edition scores ≥85 in 3 domains (cognitive, language, and motor development) with cooled infants who did not have available cells. Results Twenty-three infants were cooled and received cells. Median collection and infusion volumes were 36 and 4.3 mL. Vital signs including oxygen saturation were similar before and after infusions in the first 48 postnatal hours. Cell recipients and concurrent cooled infants had similar hospital outcomes. Thirteen of 18 (74%) cell recipients and 19 of 46 (41%) concurrent cooled infants with known 1-year outcomes survived with scores >85. Conclusions Collection, preparation, and infusion of fresh autologous UCB cells for use in infants with HIE is feasible. A randomized double-blind study is needed.

Purpose Allogeneic transplantation is typically limited to younger patients having a matched donor. To allow a donor to be found for nearly all patients, we have used a nonmyeloablative conditioning regimen in conjunction with stem cells from a related donor with one fully mismatched HLA haplotype. Patients and Methods Fludarabine, cyclophosphamide, and alemtuzumab were used as the preparatory regimen. Additional graft-versus-host disease (GVHD) prophylaxis included mycophenolate with or without cyclosporine. Patients with persistence of disease had a donor lymphocyte boost planned. Toxicities, engraftment, response, survival, and immune recovery are reported. Results Forty-nine patients with hematologic malignancies or marrow failure and no other available donors were enrolled. Ninety-four percent of patients had successful engraftment, and 8% had secondary graft failure. The treatment-related mortality rate was 10.2%, and 8% of patients had severe GVHD. Encouraging evidence of quantitative lymphocyte recovery through expansion of transplanted T cells was noted by 3 to 6 months. Seventy-five percent of patients attained a complete remission, and 1-year survival rate was 31% (95% CI, 18% to 44%). A standard-risk group of 19 patients with aplasia or in remission at transplantation demonstrated a 63% 1-year survival rate (95% CI, 38% to 80%) and 2.9-year median overall survival time (95% CI, 6.2 to 48 months). Conclusion Nonmyeloablative therapy using haploidentical family member donors is feasible because the main obstacles of GVHD and graft rejection are manageable, allowing readily available stem-cell donors to be found for nearly all patients. Further qualitative and quantitative improvement in immune recovery is needed to address the high rate of relapse and risk of severe infections.

Background: Cerebral palsy (CP) results from in utero or perinatal injury to the developing brain, often through stroke, hypoxic insult, or hemorrhage. Current treatments are supportive, focusing on managing sequelae with physical therapies, medications, and surgery. However, there are no therapies to address the underlying brain injury. Umbilical cord blood (CB) has been shown to prevent neurologic damage in children with leukodystrophiesand to improve motor function in animal models of brain injury and CP. Previously, we demonstrated safety of intravenous autologous CB infusion in young children with brain injuries. The Cerebral Palsy-Autologous Cord Blood (CP-AC) study was designed to assess the efficacy of a single intravenous infusion of autologous CB in young children with spastic CP. Methods: The CP-AC study is a prospective, randomized, double blind, placebo controlled crossover study of a single intravenous infusion of autologous CB in children ages 1-6 years with spastic CP. The Gross Motor Classification System (GMFCS) was utilized to classify the level of motor function at study entry and follow-up. Children were eligible if they were (1) GMFCS level 2-4 or (2) GMFCS level 1 with hemiplegia if they used their affected hand as an assist only. Children with known genetic conditions, intractable seizures, or severe microcephaly were ineligible. Autologous CB units had to have a documented pre-cyropreservation total cell dose of ≥1x107/kg, negative sterility culture, negative maternal infectious disease screening, and confirmed identity through HLA typing of the subject and a segment attached to the CB unit. Subjects were evaluated at baseline, one year, and two years with functional evaluations (Gross Motor Function Measure-66 (GMGM-66), Peabody Developmental Motor Scales-2, Assisting Hand Assessment, Bayley Scales of Infant Development) and brain MRI. They were randomized to the order in which they received CB and placebo infusions (given one year apart). The primary endpoint was change in GMFM-66 score at one year after the initial infusion (CB or placebo). Cryopreserved CB units were shipped to and stored at Duke until the day of treatment when they were thawed and washed in dextran 40 +5% human serum albumin (DA). Infusions, dosed at 1-5x107/kg based on the pre-cryopreservation total nucleated cell count (TNCC) and diluted in 1.25mL/kg of DA, were administered intravenously over 5-10 minutes in the outpatient setting after premedication with oral acetaminophen, IV Benadryl, and IV Solumedrol. Subjects received IV fluids and were monitored for 2-4 hours post-infusion. Results: Sixty-three children were enrolled with a median age of 2 years (range 1- 6) at baseline. Median TNCC of CB infusion was 2x107/kg (range 0.4-5) with a median CD34 dose of 0.5x105/kg (range 0.05-4). Despite negative pre-cryopreservation cultures, one CB unit grew β-hemolytic strepfrom a sample of the thawed CB unit. There were no clinical infections in this or any other study patient. One subject had transient infusion reactions consisting of hives +/- low-grade fever after each infusion; an additional dose of Benadryl was administered after the first reaction. Preliminary analysis of the 63 patients at one year showed no statistically significant overall difference in GMFM-66 change scores between placebo and treated groups (6.9 vs. 7.5, p =0.72). However, treated subjects who received pre-cryopreservation cell doses of >2.5x107/kg demonstrated statistically significant improvement in GMFM-66 change scores compared to subjects who received lower cell doses (p Conclusions: When adequately dosed, cord blood may improve motor function in young children with spastic CP. We observed improvement at pre-cryopreservation cell doses (>2.5x107/kg) that correspond with the minimum cell dose used for hematopoietic reconstitution in patients undergoing allogeneic CB transplantation. In order to extend this therapy to children who do not have adequate autologous CB units available, safety and efficacy of allogeneic CB products should be investigated. Figure 1. GMFM-66 Change Scores by Infused Cell Dose Figure 1. GMFM-66 Change Scores by Infused Cell Dose Disclosures No relevant conflicts of interest to declare.

An 8-month-old girl who seemed certain to die from the infantile form of hypophosphatasia, an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP), underwent the first trial of bone marrow cell transplantation for this heritable type of rickets. After cytoreduction, she was given T-cell-depleted, haplo-identical marrow from her healthy sister. Chimerism in peripheral blood and bone marrow became 100% donor. Three months later, she was clinically improved, with considerable healing of rickets and generalized skeletal remineralization. However, 6 months post-transplantation, worsening skeletal disease recurred, with partial return of host hematopoiesis. At the age of 21 months, without additional chemotherapy or immunosuppressive treatment, she received a boost of donor marrow cells expanded ex vivo to enrich for stromal cells. Significant, prolonged clinical and radiographic improvement followed soon after. Nevertheless, biochemical features of hypophosphatasia have remained unchanged to date. Skeletal biopsy specimens were not performed. Now, at 6 years of age, she is intelligent and ambulatory but remains small. Among several hypotheses for our patient's survival and progress, the most plausible seems to be the transient and long-term engraftment of sufficient numbers of donor marrow mesenchymal cells, forming functional osteoblasts and perhaps chondrocytes, to ameliorate her skeletal disease.

Background: Unrelated donor umbilical cord blood is an acceptable graft source for patients lacking related donors. However, a non-engraftment rate of approximately 20% despite adequate total nucleated cell (TNC) dose remains a barrier to the overall success of UCBT. Of various patient and graft characteristics that may influence engraftment, identifying an assay predictive of cord blood unit (CBU) potency and overall engraftment would be beneficial. Methods: Pre-cryopreservation (pre-cryo) and post-thaw graft characteristics were available on 423 UCBT performed at our institution between 2/11/2000 and 5/1/2007. The units were obtained from 16 US public cord blood banks and were selected by pre-cryo cell dose and HLA matching. Pre-cryo data (TNC, CD34 cells and CFU) was provided by the cord blood bank as part of routine banking practices. All units were thawed in the Duke Stem Cell Laboratory (SCL). Post-thaw testing (TNC, CD34, CFU) was performed by consistent personnel in the SCL after thaw and washing with Dextran/Albumin as described previously by Rubinstein et al. Univariate and multivariate analyses were performed to identify significant pre-cryo, post-thaw, and baseline factors predictive of neutrophil and platelet engraftment. Results: Of the 423 evaluable patients, 68% had malignancies, 61% were males, 73% were Caucasian and 38% were CMV+. The grafts were HLA (93%), sex (50%) or racially (24%) mismatched with the patients. There was excellent correlation between pre-cryo and post-thaw TNC (r2=0.92) and CD34 (r2=0.68) content, but much weaker correlation for CFUs (r2=0.27). In univariate analysis, age (≤5 years), disease (non-malignant), weight (≤12 kg), CMV status (negative), recipient ethnicity (Caucasian), HLA match (5/6 or 6/6) and pre-cryo/post-thaw TNC (larger), pre-cryo/post-thaw CD34 (larger) and pre-cryo/post-thaw CFU (larger) were predictive of both neutrophil and platelet engraftment. Multivariate analysis of parameters are presented in Table 1. In the overall multivariate analysis of neutrophil engraftment, Male units (p=0.01), 5/6 or 6/6 HLA match (p=0.02), larger post-thaw CD34 (p=0.02) and larger post-thaw CFU ( Conclusions: Post-thaw CFUs are a strong independent predictor of neutrophil and platelet engraftment after UCBT. This assay could be tested on a CBU segment and used as a marker of potency for graft selection. Factors Predictive of Neutrophil and Platelet Engraftment in Multivariate Analysis of Graft/Recipient Characteristics. Neutrophil Engraftment Platelet Engraftment Pre−Cryopreservation Multivariate Model (p−value) CD34+ (0.0046), Recipient CMV (0.0138), CFU (0.0337), Unit Sex (0.0393) Recipient ethnicity (0.0052), TNC (0.0173), CFU (0.0324) Post−Thaw Multivariate Model (p−value) CFU (

A single umbilical cord blood unit (CBU) often doses patients at the threshhold for successful hematopoietic cell transplantation. While both cryopreserved and infused total nucleated cell (TNC) content is somewhat predictive of outcomes, there has been no reliable "potency" assay developed for UCB. Frequently, pre-cryopreserved and post-thaw recoveries are inconsistent and unpredictable. The lack of standardization from cord blood bank to bank and transplant center laboratory to laboratory further complicates interpretation of the data that is currently available. Our program is in the unique position to operate both a public cord blood bank, "The Carolinas Cord Blood Bank" (CCBB) and adult and pediatric blood and marrow transplant programs performing >100 unrelated donor cord blood transplants (UCBT) per year, placing us in the unique position of evaluating cord blood pre-cryopreservation and post-thaw TNC, viability, CD34 and colony forming assays (CFU-GM, CFU-GEMM, BFU-E) in the same laboratory, eliminating the potential for differences in laboratory practices. We have evaluated the results of 218 transplants performed from CBUs obtained from the CCBB and transplanted at our center. These units did not require transfer from the bank to the transplant center (TC) eliminating the possibility that warming of CBUs during shipping could compromise cell potency. In this dataset, the cummulative incidence (CINC) of neutrophil engraftment (ANC 500/uL by day +42) was 79.4% (95% CI 74–84.8) and platelet engraftment (PLT 50K by day 180) was 61.3% (95% CI 54.8–67.8). The CINC of event-free survival (EFS) at day 180 and 1 year was 68.7% (95% CI 62.5–75) and 62.5% (95% CI 56–69.1), respectively. The CI of acute grades II–IV graft-versus-host disease at days 100 and 150 was 10.6% (95% CI 6.5–14.7%). The CI of relapse at 1 year was 8.1% (95% CI 4.4–11.8%). Thus, most deaths occurred from graft failure, infection or regimen related toxicity all of which could be influenced by graft potency. We also expanded our dataset to include an additional 405 transplants where the CBU was shipped from other cord blood banks and transplanted at Duke. In this group, the CINC of neutrophil engraftment was 78.2% (95% CI 75–81.2) and the CI of EFS at day 180 and 1 year was 60–2 (95% CI 56.3–64) and 51% (95% CI 47–55), respectively. We hypothesized that post-thaw CFU and or CD34 would be better predictors of engraftment and survival than TNC. We examined the impact of post-thaw total CFU and CD34 recoveries and dosing on neutrophil engraftment and overall survival in these patients. Dosing of post-thaw CFU/kg infused (median 3.5x10e4/kg) correlated with neutrophil engraftment (p=