Andrew E. Balber

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.

Simultaneous isolation of human BM hematopoietic, endothelial and mesenchymal progenitor cells by flow sorting based on aldehyde dehydrogenase activity: implications for cell therapy

BACKGROUND ALDH(br) cells express high aldehyde dehydrogenase (ALDH) activity and have progenitor cell activity in several contexts. We characterized human BM ALDH(br) cells to determine whether cell sorting based on ALDH activity isolates potentially useful populations for cell therapy. METHOD We measured the expression of ALDH and cell-surface Ag by flow cytometry and compared the ability of sorted ALDH(br), and BM populations remaining after ALDH(br) cells were removed (ALDH(dim) populations), to develop into several cell lineages in culture. RESULTS The ALDH(br) population comprised 1.2+/-0.8% (mean+/-SD, n=30) nucleated cells and was enriched in cells expressing CD34, CD117, CD105, CD127, CD133 and CD166, and in primitive CD34(+) CD38(-) and CD34(+) CD133(+) progenitors. Most of the CD34(+) and CD133(+) cells were ALDH(dim). ALDH(br) populations had 144-fold more hematopoietic colony-forming activity than ALDH(dim) cells and included all megakaryocyte progenitors. ALDH(br) populations readily established endothelial cell monolayers in cultures. Cells generating endothelial colonies in 7 days were 435-fold more frequent in ALDH(br) than ALDH(dim) populations. CFU-F were 9.5-fold more frequent in ALDH(br) than ALDH(dim) cells, and ALDH(br) cells gave rise to multipotential mesenchymal cell cultures that could be driven to develop into adipocytes, osteoblasts and chondrocytes. DISCUSSION Hematopoietic, endothelial and mesenchymal progenitor cells can be isolated simultaneously from human BM by cell sorting based on ALDH activity. BM ALDH(br) populations may be useful in several cell therapy applications.

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

Summary. 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™‐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.

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. STEM Cells 2011;29:570–575

Molecular cloning of p67, a lysosomal membrane glycoprotein from Trypanosoma brucei

We have previously characterized a highly glycosylated membrane protein (p67) in Trypanosoma brucei spp that is apparently targeted to lysosomes in a developmentally regulated manner. Antibody to native p67 identified a partial cDNA clone from a T. b. rhodesiense expression library and RT-PCR was used to complete the sequence of the cDNA. Equal levels of p67 transcript are detected in both procyclic and bloodstream stages of the life cycle. The 2771 nt cDNA contains a 1980 nt orf encoding a 659 amino acid polypeptide (72,567 Da). Hydropathy analysis predicts a Type I membrane topology (N to C): an N-terminal signal sequence, a large hydrophilic lumenal domain with 14 N-glycosylation sites, a trans-membrane domain (19 aa), and a short (24 aa) cytoplasmic domain. Peptide microsequencing of purified p67 identified nine residues identical to the deduced amino acid sequence, confirming the identity of the cDNA and defining the signal sequence cleavage site. Antibody to p67 protein produced in E. coli recognizes the same spectrum of native p67 glycoforms as the antibody used to clone the cDNA. All features of the deduced amino acid sequence are consistent with the known properties of the native protein and suggest a structure similar to mammalian LAMPS. The cytoplasmic domain contains two putative di-leucine targeting motifs similar to those involved in lysosomal targeting in vertebrate cells. Our results suggest that a single p67 polypeptide, or a group of highly related polypeptides, is synthesized in both bloodstream and procyclic trypanosomes and that subsequent post-translational processing and lysosomal targeting is subject to stage-specific regulation.

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

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.

Isolation of early hematopoietic cells, including megakaryocyte progenitors, in the ALDH-bright cell population of cryopreserved, banked UC blood

BACKGROUND ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors, whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS Frozen UCB units were thawed, washed, immunomagnetically depleted of cells expressing glycophorin A and CD14, reacted for flow cytometric detection of ALDH, and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H, 10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION ALDH(br) populations recovered from thawed, banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid, myeloid and megakaryocytic blood elements.

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.

PROCESSING AND TRANSPORT OF A LYSOSOMAL MEMBRANE GLYCOPROTEIN IS DEVELOPMENTALLY REGULATED IN AFRICAN TRYPANOSOMES

We have used pulse-chase immunoprecipitations methods to study early post-translational processing of CBI-gp, a lysosomal membrane glycoprotein expressed by African trypanosomes, Rap67, a polyclonal antibody to CBI-gp, immunoprecipitated a 100-kDa glycoprotein, gp100, from both bloodstream forms (BF) and procyclic forms (PF) of Trypanosoma brucei gambiense immediately after a 5-min pulse with radiomethionine. N-Glycanase digestion released a 67-kDa core protein, p67, from gp100 of both life cycle forms V8 protease digestion of p67 from BF and PF yielded 13 identical methionyl peptides, suggesting that gp100 from both life cycle forms have very similar or identical p67 core molecules. In BF, gp 100 carried both endoglycosidase H (EndoH)-resistant and EndoH-sensitive, N-linked oligosaccharides immediately after labeling. In PF, all the N-linked sugars on gp100 were EndoH sensitive. In BF, gp100 chased progressively into slower migrating 150-180-kDa components that obtained the CBI epitope, traveled to the cell surface where they could be biotinylated, and were proteolytically processed. The increase in mass of gp100 during chase in BF resulted from an elongation of N-linked oligosaccharides. Maturation of gp100 into 150-180-kDa CBI-gp was inhibited if BF were chased in the presence of glucosidase inhibitors castanospermine or deoxynojirimycin. In PF, gp100 did not increase in mass, could not be biotinylated on the cell surface, and was not proetolyzed during extended chases. Cryoimmunoelectron microscopy revealed that the antigens detected by rap67 are abundant in lysosomes and endosomes in both BF and PF. Thus, BF and PF express very similar or identical lysosomal membrane glycoproteins but process and transport them in very different ways.

A comparative study of d-lactate, l-lactate and glycerol formation by four species of Leishmania and by Trypanosoma lewisi and Trypanosoma brucei gambiense

Leishmania braziliensis panamensis, L. donovani, L. major, and L. mexicana amazonensis promastigotes, Trypanosoma lewisi bloodstream forms, and T. brucei gambiense procyclic forms were incubated with glucose as sole carbon source. All species consumed glucose more rapidly under aerobic than anaerobic conditions. All produced glycerol under anaerobic conditions, though the rate of glycerol production by T. lewisi was markedly lower than that by the other species. The four Leishmania species produced D-lactate, but not L-lactate, whereas T. b. gambiense procyclic forms produced L-lactate, but not D-lactate, and T. lewisi produced both isomers.