Human umbilical cord blood monocytes, but not adult blood monocytes, rescue brain cells from hypoxic-ischemic injury: Mechanistic and therapeutic implications.

Cord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC.

Gene products promoting remyelination are up-regulated in a cell therapy product manufactured from banked human cord blood.

BACKGROUND AIMS: DUOC-01, a cell product being developed to treat demyelinating conditions, is composed of macrophages that arise from CD14+ monocytes in the mononuclear cell (MNC) population of banked cord blood (CB). This article demonstrates that expression of multiple gene products that promote remyelination is rapidly up-regulated during manufacturing of DUOC-01 from either MNC or purified CB CD14+ monocytes. METHODS: Cell cultures were initiated with MNC or with immunoselected CD14+ monocytes isolated from the same CB unit. Cell products present in these cultures after 2 and 3 weeks were compared by three methods. First, quantitative polymerase chain reaction was used to compare expression of 77 transcripts previously shown to be differentially expressed by freshly isolated, uncultured CB CD14+ monocytes and DUOC-01. Second, accumulation of 16 soluble proteins in the culture medium was measured by Bioplex methods. Third, whole transcriptomes of the cell products were compared by microarray analysis. RESULTS: Key transcripts in multiple pathways that promote remyelination were up-regulated in DUOC-01, and substantial secretion of proteins corresponding to many of these transcripts was detected. Cell products manufactured from MNC or from CD14+ monocytes were similar with regard to all metrics. Upregulation of gene products characteristic of DUOC-01 was largely completed within 14 days of culture. CONCLUSION: We demonstrate that expression of multiple gene products that promote remyelination is up-regulated during the first 2 weeks of manufacturing of DUOC-01. Measuring these mechanistically important transcripts and proteins will be useful in monitoring manufacturing, evaluating manufacturing changes, and developing mechanism-based product potency assays.

A cord blood monocyte-derived cell therapy product accelerates brain remyelination.

Microglia and monocytes play important roles in regulating brain remyelination. We developed DUOC-01, a cell therapy product intended for treatment of demyelinating diseases, from banked human umbilical cord blood (CB) mononuclear cells. Immunodepletion and selection studies demonstrated that DUOC-01 cells are derived from CB CD14+ monocytes. We compared the ability of freshly isolated CB CD14+ monocytes and DUOC-01 cells to accelerate remyelination of the brains of NOD/SCID/IL2Rγnull mice following cuprizone feeding-mediated demyelination. The corpus callosum of mice intracranially injected with DUOC-01 showed enhanced myelination, a higher proportion of fully myelinated axons, decreased gliosis and cellular infiltration, and more proliferating oligodendrocyte lineage cells than those of mice receiving excipient. Uncultured CB CD14+ monocytes also accelerated remyelination, but to a significantly lesser extent than DUOC-01 cells. Microarray analysis, quantitative PCR studies, Western blotting, and flow cytometry demonstrated that expression of factors that promote remyelination including PDGF-AA, stem cell factor, IGF1, MMP9, MMP12, and triggering receptor expressed on myeloid cells 2 were upregulated in DUOC-01 compared to CB CD14+ monocytes. Collectively, our results show that DUOC-01 accelerates brain remyelination by multiple mechanisms and could be beneficial in treating demyelinating conditions.

Development and validation of a rapid, aldehyde dehydrogenase bright-based cord blood potency assay.

Banked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH bright [ALDH(br)]), along with viable CD45(+) or CD34(+) cell content. These measurements are made on a segment that was attached to a cryopreserved CBU. We validated the assay with prespecified criteria testing accuracy, specificity, repeatability, intermediate precision, and linearity. We then prospectively examined the correlations among ALDH(br), CD34(+), and CFU content of 3908 segments over a 5-year period. ALDH(br) (r = 0.78; 95% confidence interval [CI], 0.76-0.79), but not CD34(+) (r = 0.25; 95% CI, 0.22-0.28), was strongly correlated with CFU content as well as ALDH(br) content of the CBU. These results suggest that the ALDH(br) segment assay (based on unit characteristics measured before release) is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation.

Reprint of: Preclinical characterization of DUOC-01, a cell therapy product derived from banked umbilical cord blood for use as an adjuvant to umbilical cord blood transplantation for treatment of inherited metabolic diseases.

BACKGROUND AIMS: Cord blood (CB) transplantation slows neurodegeneration during certain inherited metabolic diseases. However, the number of donor cells in the brain of patients does not appear to be sufficient to provide benefit until several months after transplant. We developed the cell product DUOC-01 to provide therapeutic effects in the early post-transplant period. METHODS: DUOC-01 cultures initiated from banked CB units were characterized by use of time-lapse photomicroscopy during the 21-day manufacturing process. Antigen expression was measured by means of flow cytometry and immunocytochemistry; transcripts for cytokines and enzymes by quantitative real-time polymerase chain reaction; activities of lysosomal enzymes by direct biochemical analysis; alloreactivity of DUOC-01 and of peripheral blood (PB) mononuclear cells (MNC) to DUOC-01 by mixed lymphocyte culture methods; and cytokine secretion by Bioplex assays. RESULTS: DUOC-01 cultures contained highly active, attached, motile, slowly proliferating cells that expressed common (cluster of differentiation [CD]11b, CD14 and Iba1), M1 type (CD16, inducible nitric oxide synthase), and M2-type (CD163, CD206) macrophage or microglia markers. Activities of 11 disease-relevant lysosomal enzymes in DUOC-01 products were similar to those of normal PB cells. All DUOC-01 products secreted interleukin (IL)-6 and IL-10. Accumulation of transforming growth factor-ß, IL-1ß, interferon-γ and TNF-α in supernatants was variable. IL-12, IL-2, IL-4, IL-5 and IL-13 were not detected at significant concentrations. Galactocerebrosidase, transforming growth factor-ß and IL-10 transcripts were specifically enriched in DUOC-01 relative to CB cells. PB MNCs proliferated and released cytokines in response to DUOC-01. DUOC-01 did not proliferate in response to mismatched MNC. CONCLUSIONS: DUOC-01 has potential as an adjunctive cell therapy to myeloablative CB transplant for treatment of inherited metabolic diseases.

Preclinical characterization of DUOC-01, a cell therapy product derived from banked umbilical cord blood for use as an adjuvant to umbilical cord blood transplantation for treatment of inherited metabolic diseases.

BACKGROUND AIMS: Cord blood (CB) transplantation slows neurodegeneration during certain inherited metabolic diseases. However, the number of donor cells in the brain of patients does not appear to be sufficient to provide benefit until several months after transplant. We developed the cell product DUOC-01 to provide therapeutic effects in the early post-transplant period. METHODS: DUOC-01 cultures initiated from banked CB units were characterized by use of time-lapse photomicroscopy during the 21-day manufacturing process. Antigen expression was measured by means of flow cytometry and immunocytochemistry; transcripts for cytokines and enzymes by quantitative real-time polymerase chain reaction; activities of lysosomal enzymes by direct biochemical analysis; alloreactivity of DUOC-01 and of peripheral blood (PB) mononuclear cells (MNC) to DUOC-01 by mixed lymphocyte culture methods; and cytokine secretion by Bioplex assays. RESULTS: DUOC-01 cultures contained highly active, attached, motile, slowly proliferating cells that expressed common (cluster of differentiation [CD]11b, CD14 and Iba1), M1 type (CD16, inducible nitric oxide synthase), and M2-type (CD163, CD206) macrophage or microglia markers. Activities of 11 disease-relevant lysosomal enzymes in DUOC-01 products were similar to those of normal PB cells. All DUOC-01 products secreted interleukin (IL)-6 and IL-10. Accumulation of transforming growth factor-ß, IL-1ß, interferon-γ and TNF-α in supernatants was variable. IL-12, IL-2, IL-4, IL-5 and IL-13 were not detected at significant concentrations. Galactocerebrosidase, transforming growth factor-ß and IL-10 transcripts were specifically enriched in DUOC-01 relative to CB cells. PB MNCs proliferated and released cytokines in response to DUOC-01. DUOC-01 did not proliferate in response to mismatched MNC. CONCLUSIONS: DUOC-01 has potential as an adjunctive cell therapy to myeloablative CB transplant for treatment of inherited metabolic diseases.

Development of a flow cytometry-based pulse-width assay for detection of aggregates in cellular therapeutics to be infused by catheter.

BACKGROUND AIMS: Delivery of cell-based therapies through the carotid artery with the use of an intra-arterial catheter could introduce aggregates and cause focal ischemia in the brain. We developed a pulse-width flow cytometry method for aggregate detection and quantification. The assay was designed to be used as a cell product release assay in a clinical trial seeking to treat ischemic stroke with sorted cells brightly expressing aldehyde dehydrogenase (ALDH(br) cells) delivered through intra-arterial catheters. METHODS: The forward light scatter pulse-width axis of a flow cytometer was calibrated for particle diameter measurements through the use of traceable standard microspheres and linear regression. As a positive control, Concanavalin A-aggregated cells were counted manually and sorted onto slides to compare with pulse width-determined values. Known numbers of aggregates were spiked into purified singlet cells for quantification. A clinical standard for aggregate count and diameter was determined. The assay was used to qualify catheters with the use of ALDH(br) cells. RESULTS: The pulse-width axis was highly linear for microsphere diameter (r(2) > 0.99), which allowed for size calibration. Microscopically determined counts and diameters corresponded to pulse width-determined values. Known aggregate counts were linear with pulse width-determined aggregate counts (r(2) = 0.98). The limit of detection was determined to be 0.004%. Flow of ALDH(br) cells through catheters did not generate aggregates. The final method to be used as a release assay for the stroke clinical trial was tested successfully on samples from volunteer donors. CONCLUSIONS: The pulse-width aggregate detection assay provides a reliable, reproducible, accurate and rapid means of detection, classification and quantification of aggregates in cell therapy products.

Optimizing donor selection for public cord blood banking: influence of maternal, infant, and collection characteristics on cord blood unit quality.

BACKGROUND: Banked unrelated donor umbilical cord blood (CB) has improved access to hematopoietic stem cell transplantation for patients without a suitably matched donor. In a resource-limited environment, ensuring that the public inventory is enriched with high-quality cord blood units (CBUs) addressing the needs of a diverse group of patients is a priority. Identification of donor characteristics correlating with higher CBU quality could guide operational strategies to increase the yield of banked high-quality CBUs. STUDY DESIGN AND METHODS: Characteristics of 5267 CBUs donated to the Carolinas Cord Blood Bank, a public bank participating in the National Cord Blood Inventory, were retrospectively analyzed. Eligible CBUs, collected by trained personnel, were processed using standard procedures. Routine quality and potency metrics (postprocessing total nucleated cell count [post-TNCC], CD34+, colony-forming units [CFUs]) were correlated with maternal, infant, and collection characteristics. RESULTS: High-quality CBUs were defined as those with higher post-TNCC (>1.25 × 10(9)) with CD34+ and CFUs in the upper quartile. Factors associated with higher CD34+ or CFU content included a shorter interval from collection to processing (<10 hr), younger gestational age (34-37 weeks; CD34+ and CFUs), Caucasian race, higher birthweight (>3500 g), and larger collection volumes (>80 mL). CONCLUSIONS: We describe characteristics identifying high-quality CBUs, which can be used to inform strategies for CBU collection for public banks. Efforts should be made to prioritize collections from larger babies born before 38 weeks of gestation. CBUs should be rapidly transported to the processing laboratory. The lower quality of CBUs from non-Caucasian donors highlights the challenges of building a racially diverse public CB inventory.

The Cord Blood Apgar: a novel scoring system to optimize selection of banked cord blood grafts for transplantation (CME).

BACKGROUND: Engraftment failure and delays, likely due to diminished cord blood unit (CBU) potency, remain major barriers to the overall success of unrelated umbilical cord blood transplantation (UCBT). To address this problem, we developed and retrospectively validated a novel scoring system, the Cord Blood Apgar (CBA), which is predictive of engraftment after UCBT. STUDY DESIGN AND METHODS: In a single-center retrospective study, utilizing a database of 435 consecutive single cord myeloablative UCBTs performed between January 1, 2000, to December 31, 2008, precryopreservation and postthaw graft variables (total nucleated cell, CD34+, colony-forming units, mononuclear cell content, and volume) were initially correlated with neutrophil engraftment. Subsequently, based on the magnitude of hazard ratios (HRs) in univariate analysis, a weighted scoring system to predict CBU potency was developed using a randomly selected training data set and internally validated on the remaining data set. RESULTS: The CBA assigns transplanted CBUs three scores: a precryopreservation score (PCS), a postthaw score (PTS), and a composite score (CS), which incorporates the PCS and PTS values. CBA-PCS scores, which could be used for initial unit selection, were predictive of neutrophil (CBA-PCS ≥ 7.75 vs. <7.75, HR 3.5; p < 0.0001) engraftment. Likewise, CBA-PTS and CS scores were strongly predictive of Day 42 neutrophil engraftment (CBA-PTS ≥ 9.5 vs. <9.5, HR 3.16, p < 0.0001; CBA-CS ≥ 17.75 vs. <17.75, HR 4.01, p < 0.0001). CONCLUSION: The CBA is strongly predictive of engraftment after UCBT and shows promise for optimizing screening of CBU donors for transplantation. In the future, a segment could be assayed for the PTS score providing data to apply the CS for final CBU selection.

Total colony-forming units are a strong, independent predictor of neutrophil and platelet engraftment after unrelated umbilical cord blood transplantation: a single-center analysis of 435 cord blood transplants.

Graft failure occurs in approximately 20% of patients after unrelated umbilical cord blood transplantation (UCBT). This could be because of inadequate potency of the cord blood unit (CBU). To this end, we investigated the impact of graft characteristics on engraftment and survival of 435 primarily pediatric (median age: 5.3 years) patients receiving a single-unit unrelated UCBT after myeloablative conditioning from 2000 to 2008. Pre-cryopreservation (pre-cryo) graft characteristics were provided by the banks. Post-thaw parameters were measured on dextran/albumin-washed grafts. Post-thaw recovery of the colony-forming unit (CFU), a biological assay reflecting functional viability of the cord blood cells was the lowest percent age (median 21.2%, mean 36.5%) of the pre-cryo value, regardless of the bank of origin. The cumulative incidences of neutrophil and platelet engraftment were 76.9% (95%, confidence interval [CI], 71.3%-82.5%) and 55% (95% CI, 49.3%-60.7%), respectively. Univariate and separate multivariate models using pre-cryo and post-thaw datasets including clinical parameters identified predictors of engraftment and survival. In multivariate modeling, higher CFU dosing was the only pre-cryo graft characteristic predictive of neutrophil (P = .0024) and platelet engraftment (P = .0063). In the post-thaw model, CFU dose best predicted neutrophil and platelet engraftment (both P < .0001). Comparatively, CD34(+) and total nucleated cell (TNC) were only weakly predictive in post-thaw neutrophil and platelet engraftment models, respectively. In conclusion, CFU dose is a strong independent predictor of engraftment after unrelated UCBT and should be used to assess potency when selecting CBUs for transplantation.

Concise review: aldehyde dehydrogenase bright stem and progenitor cell populations from normal tissues: characteristics, activities, and emerging uses in regenerative medicine.

Flow cytometry has been used to detect cells that express high levels of the aldehyde dehydrogenase activity in normal tissues. Such ALDH bright (ALDHbr) cell populations have been sorted from human cord blood, bone marrow, mobilized peripheral blood, skeletal muscle, and breast tissue and from the rodent brain, pancreas, and prostate. A variety of hematopoietic, endothelial, and mutiltipotential mesenchymal progenitors are enriched in the human bone marrow, cord, and peripheral blood ALDHbr populations. Multipotential neural progenitors are enriched in rodent brain tissue, and tissue-specific progenitors in the other tissue types. In xenograft models, uncultured human bone marrow and cord ALDHbr cells home to damaged tissue and protect mice against acute ischemic injury by promoting angiogenesis. Uncultured cord ALDHbr cells also deploy to nonhematopoietic tissues and protect animals in CCl4 intoxication and chronic multiorgan failure models. Mouse ALDHbr cells and cells derived from them in culture protect animals in a chronic neurodegenerative disease model. Purifying ALDHbr cells appears to increase their ability to repair tissues in these animal models. Clinical studies suggest that the number of ALDHbr cells present in hematopoietic grafts or circulating in the blood of cardiovascular disease patients is related to clinical outcomes or disease severity. ALDHbr cells have been used to supplement unrelated cord blood transplant and to treat patients with ischemic heart failure and critical limb ischemia. ALDH activity can play several physiological roles in stem and progenitor cells that may potentiate their utility in cell therapy.

Mobilized peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation.

We have developed an approach for identifying primitive mobilized peripheral blood cells (PBSC) that express high levels of aldehyde dehydrogenase (ALDH). PBSC were stained with a fluorescent ALDH substrate, termed BODIPY trade mark -aminoacetaldehyde (BAAA), and then analysed using flow cytometry. A population of cells with a low side scatter (SSC) and a high level of BAAA staining, termed the SSCloALDHbr population, was readily discriminated and comprised a mean of 3 +/- 5% of leukapheresis samples. A mean of 73 +/- 11% of the SSCloALDHbr population expressed CD34 and 56 +/- 25% of all the mobilized CD34+ cells resided within the SSCloALDHbr population. The SSCloALDHbr population was largely depleted of cells with mature phenotypes and enriched for cells with immature phenotypes. Sorted SSCloALDHbr and SSCloALDHbr CD34+ PBSC were enriched for progenitors with the ability to (1) generate colony-forming units (CFU) and long-term culture (LTC)-derived CFU, (2) expand in primary and secondary LTC, and (3) generate multiple cell lineages. In 21 cancer patients who had undergone autologous PBSC transplantation, the number of infused SSCloALDHbr cells/kg highly correlated with the time to neutrophil and platelet engraftment (P < 0.015 and P < 0.003 respectively). In summary, peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation.

Developmentally regulated trafficking of the lysosomal membrane protein p67 in Trypanosoma brucei.

p67 is a lysosomal type I membrane glycoprotein of Trypanosoma brucei. In procyclic stage cells p67 trafficks to the lysosome without modification, but in the bloodstream stage Golgi processing adds poly-N-acetyllactosamine to N-glycans. In both stages proteolytic fragmentation occurs in the lysosome, but turnover is approximately nine times faster in bloodstream cells. Trafficking of wildtype p67 and mutants missing the cytoplasmic (p67DeltaCD) or cytoplasmic/transmembrane domains (p67DeltaTM) was monitored by pulse-chase, surface biotinylation and immunofluorescence. Overexpressed wildtype p67 trafficks normally in procyclics, but some leaks to the cell surface suggesting that the targeting machinery is saturable. p67DeltaCD and p67DeltaTM are delivered to the cell surface and secreted, respectively. The membrane/cytoplasmic domains function correctly in procyclic cells when fused to GFP indicating that these domains are sufficient for stage-specific lysosomal targeting. In contrast, p67 wildtype and deletion reporters are overwhelmingly targeted to the lysosome and degraded in bloodstream cells. These findings suggest that either redundant developmentally regulated targeting signals/machinery are operative in this stage or that the increased endocytic activity of bloodstream cells prevents export of the deletion reporters.