DUOC-01 is a cord-blood derived, macrophage-based cell therapy product, developed by our group to treat demyelinating conditions of the central nervous system. Recently, we demonstrated that DUOC-01 accelerated remyelination, decreased gliosis, and reduced cellular infiltration in the corpus callosum of immune-incompetent mice treated with cuprizone. To explore the mechanism and investigate whether DUOC-01 will be effective in other experimental models of demyelination, we tested DUOC-01 in lysophosphatidylcholine (LPC) mediated demyelinated murine organotypic cerebellar brain slices and a mouse model of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. In the cerebellar brain slices, we found that DUOC-01 enhanced remyelination, reduced gliosis, and promoted the proliferation of oligodendrocyte progenitor cells in brain slices demyelinated by LPC compared to the untreated control samples. When we injected DUOC-01 cells into the cerebrospinal fluid of mice immunized for EAE, we found decreased severity of clinical disease compared to the vehicle injected control. Currently we are analyzing single cell sequencing data to determine various populations present in DUOC-01 cultures and to understand the functional pathways responsible for promoting remyelination and reducing neuroinflammation. Overall, our data suggest that DUOC-01 could be beneficial in treating diverse neurological conditions with demyelination.

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.

Background: AMR-001, an autologous CD34+ cell product derived from mini-marrow harvest, is currently undergoing Phase II trials to treat acute myocardial infarction (AMI). At the time of AMR-001 infusion, it is believed that the infarct-region stromal derived factor-1 (SDF-1) levels are peaked. It was found that improvement in cardiac perfusion and infarct size correlated with the mobility potential of CD34+ cells, as mediated by a SDF-1 gradient. We have initiated a study to identify potential microRNAs (miRNAs) and proteins that may be used as biomarkers that correlate to CD34+ cell migratory potential. Methods: In vitro transwell migratory assays were performed on purified CD34+ cells derived from bone marrow of healthy donors. After 4 hours at 37 C, CD34+ cells that migrated into the lower chamber in the presence of SDF-1, the non-mobilized cells in the upper chamber, and untreated cells were harvested. The miRNA expression profile was analyzed (Sistemic, Ltd) using microarray slides (Agilent). A biotin label-based antibody array (RayBiotech) was used for the detection of 1000 proteins. Results: Hierarchical clustering analysis of the miRNA data showed that mobilized cells grouped separately from the non-mobilized/untreated cells. Sixty-three miRNAs were upregulated in the mobilized samples compared to non-mobilized/untreated samples, including two miRNAs which have a reported pro-angiogenic role in migratory cells. Twenty-six proteins had higher expression in mobilized cells compared to non-mobilized cells. Several of the identified proteins have a role in migration or angiogenesis. Conclusion: Analysis of the miRNA and protein profiles of the CD34+ cells identified a number of miRNAs/proteins that represent possible markers for a migratory phenotype. qPCR and ELISA assays will be performed to verify the specific miRNAs and proteins identified. This approach will enable the development of a biomarker assay for migratory potential of AMR-001.

Hypoxic-ischemic (HI) brain injury is a frequent cause of perinatal morbidity and mortality and treatment options are limited. Human umbilical cord blood (CB) mononuclear cells (MNC) are being tested in the clinic for treatment of HI brain injuries with encouraging preliminary results. However the molecular mechanism(s) that underlie this clinical effect are not understood. Recently, we have shown that CB-MNC protected brain neurons from Oxygen-Glucose-Deprivation (OGD)-induced death and suppress astrocyte activation much more effectively than MNC from adult peripheral blood (PB) and that CB-CD14+ monocytes mediated these protective activities. Here, to determine molecules that may contribute to the neuroprotective effect of CB-CD14+ cells, we analyzed the transcriptomes of CB and PB-CD14+ monocytes and found that they differed in expression of many transcripts. We identified seven of these transcripts encoded secreted proteins in CB-CD14+ that could play a paracrine role in neuroprotection. We show that supernatants conditioned by CB CD14+ monocytes exposed to factors released from OGD-shocked brain slices were also neuroprotective. Western blot analysis confirmed that CB monocytes over-expressed five of the proteins corresponding to these candidate genes. Two of these, thrombospondin 1 and chitinase 3-like protein 1, were detected in secretory granules of all CB, but not PB, monocytes by immunofluorescence. The matrix metalloproteinase 9, was abundant in a subpopulation of CB monocytes but was rare in PB monocytes. Our data suggest that CB CD14+ monocytes play a central role in the treatment of HI with CB MNC, allude to specific proteins that may participate in neuroprotection, and suggest new, mechanism-based approaches for developing cell therapy products derived from CB units for treatment of HI brain injury.

Umbilical cord tissue or Wharton's Jelly is a rich source of rapidly proliferating mesenchymal stromal cells (human cord tissue MSCs; hCTMSC) that can be cultured on a large-scale. We are developing this MSC product for use in the treatment of children with autism spectrum disorders (ASDs) where increasing evidence points to a central role for immune dysregulation involving abnormal activation of microglial cells. We developed an organotypic brain slice culture model to ask whether hCTMSC inhibit microglial activation in vitro. We plan to use the assay to explore whether it could serve as a biomarker to predict the potential therapeutic effectiveness of various lots of MSCs in patients with ASDs.

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.

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.

Approximately 87% of adult strokes are ischemic in etiology, and occur when cerebral blood flow is blocked by a clot or mechanical event. Immediately after the stroke, inflammatory mediators may exacerbate the development of cerebral edema and secondary tissue injury. Cell therapy may favorably alter the natural history of these processes through paracrine signaling that reduces inflammation, promotes angiogenesis, neurogenesis and recruitment of endogenous cell repair mechanisms. We hypothesize that cell therapy using non HLA matched umbilical cord blood (CB) from an unrelated donor would provide benefit by reducing the area of permanent injury and improving functional outcomes for these patients. The CoBIS study is an IRB approved, FDA IND sponsored, prospective, open-label, multi-center, Phase 1 safety study of a single intravenous infusion of allogeneic CB in 10 adults ages 18-80 years old. Cord blood units are selected by ethnicity, blood type, and ability to supply a dose of 0.5 – 1.5 x 107 TNCC/kg. Eligible patients include those experiencing a recent, acute cortical, hemispheric, ischemic stroke in the middle cerebral artery (MCA) distribution as detected by MRI as a diffusion weighted abnormality and were enrolled if their National Institutes of Health Stroke Scale (NIHSS) was 8-15 (right hemisphere) or 8-18 (left hemisphere). Subjects who receive tPA or undergo mechanical perfusion were eligible for inclusion. Subjects are not pre-treated with immunosuppressive drugs. Cord blood units are selected to match for ABO/Rh and race. The primary endpoint is safety as assessed by the frequency and severity of adverse events within 24 hours of cord blood infusion and 12 month period post CB infusion. Secondary outcome measures include Modified Rankin Scale (mRS), NIHSS, the Barthel Index (BI), and European Quality of Life (EQ-5D-3L), Patient Health Questionnaire Scale (PHQ8), Telephone Interview for Cognitive Status (TICS), and a self-reporting survey of rehabilitation therapy. MRI will be used to evaluate changes in the brain 3 months post infusion. To date (SEPT2015) two subjects have been enrolled at Duke University. Two males, ages 63 and 69, were infused day 8 and 9, respectively, post the initial stroke event. The dosing window for the 10 subjects is 3-10 days post stroke. At 60 days post infusion for both subjects, there have been no serious adverse events and both subjects were discharged from the hospital for rehabilitation therapy. Early safety data suggests intravenous infusion of unmatched, allogeneic, CB cells is well tolerated. If the safety profile remains favorable, we will move to a randomized, placebo controlled Phase 2 study. Our goal to use unrelated non-HLA matched CB to down-regulate inflammation and to promote neuroprotection and neurorepair in patients with ischemic stroke.

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.

Our lab is developing cord blood (CB)-derived cell therapies for neuronal damage resulting from hypoxic-ischemic [HI] insult. We are using mouse brain slice cultures subjected to oxygen-glucose deprivation [OGD] to study how CB cells mediate neuroprotection. We previously reported that CD14+ cells account for most of the neuroprotective activity of CB cells in this model. We used immunohistochemistry to further detail the mechanisms of this neuroprotection. Brain slice cultures established from C57BL/6J mice were subjected to 1h OGD on day 9 treated with cell populations or medium immediately after normal conditions were restored. CB CD14+ and CD14+ depleted cells were immunomagnetically prepared from CB mononuclear cells within 48h of collection. Human adult peripheral blood (PB) CD14+ populations were also tested. After 72h, slice cultures were fixed and stained with antibodies to detect astrocytes (GFAP), neurons (NeuN), oligodendrocytes (olig2), and microglia (Iba1). Glial and neuronal cells were enumerated in contiguous images of the periventricular regions using fluorescence confocal microscopy. We also characterized the effects of cell treatment on primary human astrocytes subjected to OGD stress in a microfluidics chamber. In both culture systems treatment with CB-CD14+ cells resulted in an increase in NeuN+ neurons and a decrease in the number of activated GFAP+ astrocytes following OGD shock. Cultures treated with CB-CD14+ had 2-fold more surviving neurons than those not treated. CD14 depleted cells did not protect cultures. We did not detect changes in microglia or oligodendrocytes following cell treatment. We conclude that CB CD14+ cells demonstrate a greater neuroprotective and anti-neuroinflammatory effect than PB CD14+ cells. CB CD14+ cells could mediate neuroprotection either directly on neurons or indirectly through modulation of astrocyte activation. We confirm the therapeutic potential of CB CD14+ cells in the setting of acquired HI.

We have developed an umbilical cord blood-derived cell product, DUOC-01, as a potential adjunct therapy to facilitate neural repair in patients with leukodystrophies. In clinical practice, DUOC-01 cells will be transplanted by intrathecal injection several weeks after the patient receives a systemic cord blood transplant. To validate this strategy, we developed a preclinical model whereby DUOC-01 cells were transplanted by intrathecal injection into neonatal (≤ 2 days old) NOD/SCID-IL2Rγnull (NSg) mice. In our prior work, we analyzed the tissue distribution of the cells using quantitative PCR to detect human Alu DNA sequences. That work demonstrated that, within the first 24 hours, the DUOC-01 cells were detectable in all mice that had been transplanted (n=5); and, localized to both neural and non-neural tissues, including the brain, spinal cord, lungs and liver. Human cells remained detectable within approximately half of all mice for periods of up to 56 days post-transplantation (n=22); however, from day 7 onward, the cells were only detectable within the brain and spine.

Delayed engraftment and graft failure are barriers to the success of unrelated donor cord blood transplantation (UCBT). This is due, in part, to decreased potency of the infused cord blood unit (CBU). We previously demonstrated that post-thaw colony forming units (CFU) is a strong predictor of survival and engraftment after UCBT. However, the usefulness of CFU is limited by time and variability of assay results. Recently Barker and colleagues showed that viability of the CD34 population of a thawed CBU product predicted the engrafting unit in double UCBT. However, an assay assessing potency of the CBU prior to final selection for transplantation would be optimal. We developed a potency assay performed on segment attached to a CBU, enumerating the content of ALDHbr [Aldecount®], CD34+, CD45+, glycophorin A+ and viability (7-AAD+), with CFUs, on CBUs requested for human leukocyte antigen confirmatory typing. From March 2010-August 2013, segments from 2766 CBUs were analyzed. The percentage of viable CD45+ cells expressing ALDHbr (r=0.82) or ALDHbr/CD34+ (r=0.72) correlated well with CFUs (n=2730, both p<0.0001). In contrast, CFU correlated less well with the percent of viable CD45+ cells expressing CD34+ (n=2727, r=0.28, p<0.0001) or the overall viability of CD34+ cells (n=2727, r=0.13, p<0.0001). In a cohort of patients with malignancies receiving a single CBU transplant after myeloablative conditioning, patients receiving cords low in ALDHbr or CFU experienced delayed engraftment compared to patients receiving patients receiving cord with higher levels of either CFUs or ALDHbr cells. In contrast, the viable CD34 content of the transplanted CBU was not a significant predictor of engraftment delay. Therefore, ALDHbr, which highly correlates with CFU, may be superior to viability of the CD34 population as a post-thaw potency measurement for CBU. Assaying the segment allows for identification of CBUs with higher potency before final selection of a CBU for UCBT.

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-β, IL-1β, 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-β 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.

We have developed an umbilical cord blood-derived cell product, DUOC-01, as a potential adjunct therapy for patients with certain inherited leukodystrophies. In clinical practice, DUOC-01 cells will be transplanted only after systemic cord blood transplantation and engraftment. Importantly, the DUOC-01 cells will be derived from the same cord blood unit that is to be used for the systemic transplant. To facilitate neural repair, the DUOC-01 cells will be delivered by intrathecal injection. The goals of this study were to determine the tissue distribution of DUOC-01 cells following their intrathecal injection, and to determine how long they remain detectable in vivo. Five different DUOC-01 cell preparations were cultured per GMP-compliant Standard Operating Procedures. For each transplant, 105 cells were delivered by intrathecal injection into neonatal (≤ 2 days old) NOD/SCID-IL2Rγnull mice. After periods of up to 56 days post-transplantation, the mice were sacrificed to analyze six tissues (brain, spinal cord, lungs, liver, spleen and bone marrow) for their content of human cells, as determined using quantitative PCR to detect human Alu DNA sequences. Within the first 24 hours post-transplantation, the DUOC-01 cells were detected within multiple tissues in 5 of 5 mice. These included the brain, spinal cord, lungs or, to a lesser degree, the liver. Human cells remained detectable within 11 of 20 mice that were analyzed between 7 and 56 days post-transplantation. However, at these later time points, the cells were detected only within the brain and spinal cord. Furthermore, in 8 mice that had human cells detectable within both the brain and spinal cord, the human DNA was more prevalent in the brain. These studies indicate that immediately following intrathecal injection the DUOC-01 cells distribute to both neural and non-neural tissues. However, in the long-term, the cells only remained detectable in the neural spaces encompassed by the brain and spinal cord.

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 cytometty 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 inva-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.

Allogeneic umbilical cord blood [CB] transplantation can slow or reverse progression of central nervous system demyelination in inherited metabolic diseases. Clinical observations suggest that several months are required after transplant for donor derived cells in the brain to provide benefit. We are developing DUOC-01 as a bridging cell product administered intrathecally to patients early post-transplant to provide therapeutic effects prior to CNS engraftment by cells from the CB transplant. DUOC-01 is manufactured under cGMP conditions from the 20% compartment of the same CB unit used for systemic transplantation by a modification of a previously described method. We performed preclinical characterization of DUOC-01. Time lapse imaging showed that the cultures evolve into attached, motile, highly active cell populations resembling macrophages. The cells express characteristic myeloid macrophage markers including CD45, CD11b, and Iba1. Cells had activities of 11 disease-relevant lysosomal enzymes similar to wild type blood leucocytes. All DUOC-01 batches secreted IL-6 and IL-10. Some secreted TGF-β, IL-1β, INF-γ, TNF-α or very low amounts of IL-12 or IL-2. IL-4, IL-5 and IL-13 were not detected. Peripheral blood mononuclear cells [MNC] proliferated and released cytokines in response to DUOC-01. CB MNC did not respond to DUOC-01 made from the same unit, and DUOC-01 did not proliferate in response to mismatched MNC. Following intrathecal injection DUOC-01 cells were targeted to and persisted in brains and spinal cords of newborn NOD/SCID-IL2Rγnull mice for up to 56 days. Brains of NOD-SCID mice injected intrathecally or intracerebrally with DUOC-01 showed no tumors, ectopic tissue growth, or gross clinical abnormalities during 56 days of observation. DUOC-01 accelerated remyelination in NOD/SCID-IL2Rγnull mouse brains following curpizone feeding. Thus, preclinical studies suggest that DUOC-01 has promise as a candidate cell therapy for demyelinating diseases.

Specifications for CD34 viability and recovery from cryopreserved and thawed umbilical cord blood have yet to be defined. Various banks, transplant centers, registries and regulatory bodies use terminology about CD34 viability or recovery of viable CD34 cells with precision. Comparability between methods or assays used to measure or calculate viable CD34 cells have not been established. Knowledge about the meaning of a viable CD34 or percent recovery of viable CD34 cell result is poor amongst transplanters and registries utilizing this data. In the US, the specifications set forth in the FDA Guidance for Cord Blood Licensure is equally confusing and based on a error (pre 2005) when validated assays for viable CD34 essentially did not exist. In those historical times, one approach to quantitation of viable CD34 cells involved measurement of the total nucleated cell count multiplied by the percent viability measured by trypan blue multiplied by the percent of CD34 cells enumerated by flow cytometry. Recent assays incorporate vital dyes, like 7-AAD, into flow cytometry panels with CD34. A viable CD34 count/uL as well as viability of CD34 cells are reported. These can be multiplied by the volume of product recovered post thaw to quantitate a total viable CD34 count. This can be divided by the pre cryopreservation viable CD34 cell content to calculate percent recovery of viable CD34 cells. These approaches yield different absolute results. Also, these assays often have a preparation step using lysis of red blood cells which also lyses some of the nucleated white blood cells in the product, a step which, along with prolongation of timing of the assay run, may reduce the measured amount of recovered viable CD34 cells. Other variations will be discussed. The transplant and banking communities need to understand what viable CD34 means and the scientific and banking communities need to a new approach to measurement of and the language used to talk about viable CD34 cells.

Our laboratory is developing novel cord blood (CB)-derived cellular therapies for patients with neuronal damage resulting from hypoxia-ischemia. To understand how CB cells mediate response to injury, we adapted and characterized the organotypic mouse brain slice culture model. The cellular composition of these brain slice cultures can be used to better understand the mechanisms underlying hypoxic injury and beneficial effects of cell therapies. We used immunofluorescence and image analysis to enumerate glial and neuronal cells in C57BL/6J mice-derived brain slices cultured on a semi-permeable membrane for 21 days in serum free medium. We compared the cellular composition of ex vivo brain slices to that of neonatal mouse brains. Selected brain slice cultures or brain sections were fixed in 4% paraformaldyde on ex-vivo culture or postnatal days 1, 3, 6, 9, 12, 15 and 21. Immunohistochemical staining differentiated astrocytes (GFAP), neurons (NeuN), oligodendrocytes (olig2) and microglia (Iba1) in cultured brain slices. Contiguous images of the periventricular regions were analyzed using fluorescence confocal microscopy. In brain slice cultures, neurons comprised approximately 40% (SD +/- 7%) of the total cell population, while glial cells made up 60% (SD +/- 3.5%) throughout the 21 day culture period. Conversely, in the age-matched neonatal brain sections, neurons maintained an average of 60% (SD +/- 6%) of the cellular composition, and remaining glial cells were 40% (SD +/- 4%). Both in vivo and in vitro, the relative proportions of the three glial cell populations were the same. These results establish concordance between the in vivo and ex vivo systems and validate the ex vivo brain slice model for use to further investigate effects of hypoxic injury. We have used this model system to investigate the protective effects of cord blood mononuclear cells after acute hypoxic injury on different types of brain cells.

We are developing a candidate cell therapy product, DUOC-01, derived from banked cord blood for use in the treatment of CNS demyelination. We have adapted the cuprizone model of reversible brain demyelination to determine whether DUOC-01 can accelerate remyelination of neurons in immune-incompetent NOD/SCID-IL2Rγnull [NSG] mice. Male, 7-9 week old NSG mice were adapted to a milled lab chow diet for one week and then shifted to chow containing 0.2% (w/w) cuprizone. After 5 weeks, brains were harvested from cuprizone-fed and from controls kept on standard chow for subsequent assessment of the degree of demyelination and gliosis induced by cuprizone. Remaining animals were returned to normal lab chow to allow remyelination to begin. One day after the change in diet, one group of mice was stereotactically injected in the corpus callosum [CC] region with 105 DUOC-01 cells. Cells were manufactured using protocols suitable for clinical use. Control mice were injected with excipient only. Brains were harvested from DUOC-01 treated and control mice 1 week following injections, and cryosections were prepared. Myelination was assessed by Luxol-fast blue-periodic acid Schiff-staining [LFB] and immunohistochemistry [MBP]. Organization of neurons [NHF] and distribution of astrocytes [GFAP] and oligodendrocytes [Oligo2] were also assessed by immunohistochemistry. The CC midline region and more lateral regions of NSG mice were severely demyelinated with gliosis following cuprizone feeding. LFB staining one week after cell treatment showed that mice injected with DUOC-01 had significantly increased myelination [p<0.0006] and decreased gliosis and cellular infiltration [p<0.01] in the CC region compared to mice injected with excipient. No abnormalities were noted in the brains of animals maintained on standard chow throughout the protocol and injected with DUOC-01 or excipient. These data demonstrate the potential activity of DUOC-01 in treating demyelinating conditions.

We are developing clinical products derived from human cord blood [CB] mononuclear cells [CBMC] to protect the brain from acute hypoxic injury. We have standardized an organotypic mouse brain slice culture model to identify CBMC subpopulations that protect brain cells from death following oxygen-glucose deprivation [OGD]. To prepare CBMC, <2day old CB units were centrifuged on Ficoll®, treated with NH4Cl, and washed in medium. Brain slice [300μm] cultures established from P1 or P2 C57BL/6J mice were maintained for 8-10 days on membrane filters over serum free medium under normoxic conditions, subjected to OGD [glucose free medium in <1 % O2; 1 hour], and then returned to normal conditions. CBMC were then added on top of the slices. Co-cultures were maintained 72h. OGD induced death was measured by propidium iodide staining. Adding CBMC reduced cell death in a dose dependent manner; 25,000 CBMC reduced cell death 80 ± 5% [mean ± SD, n=5]. Peripheral blood [PB] mononuclear cells showed 3-fold less protection. Adding 125,000 CBMC to the medium below the membrane instead of directly to slices reduced brain cell death 60 ± 8 % [mean +/-SD; n=3], suggesting that CBMC produce diffusible protective factors. To identify what types of cells mediate protection, we immunomagnetically depleted specific cell types and added depleted CBMC populations to OGD shocked cultures. Depleting CD14+ cells reduced the protective activity of CBC 3.5-fold, but depleting CD3+, CD19+, or CD34+ cells did not remove protective activity. Positively selected CD14+ also protected brain cultures from OGD, but CD3+ and CD19+ enriched populations did not. CD14+ cells selected from PB were 4-fold less active than CD14+ cells isolated from CB. Thus, CD14+ cells from CB are uniquely active in protecting brain cells from OGD induced death. We are now exploring how CB CD14+ cells interact with brain glia and neurons in these cultures and the molecular mechanisms by which CD14+ cells protect brain cells.