Separation and Further Characterization of Hematopoietic Cell Populations Based on Phenotypic and Biophysical Properties

Hematopoietic stem cells (HSC) are multipotent and possess self-renewal capacity. Thus, they can sustain a life-long hematopoiesis and are also capable of restoring the hematopoietic system when transplanted to a patient. In allogeneic hematopoietic stem cell transplantation (HSCT), a potent graft-versus-leukemia (GvL) effect must be balanced against the concomitant risk of severe immunological response to non-malignant host cells; graft-versus-host-disease (GvHD). In case of recipient-donor tissue antigen disparity, such as human leukocyte antigen (HLA)-mismatch or ABO-incompatibility, depletion of allo-reactive or incompatible blood constituents by HSCT graft processing may be required. The aim of this work was to investigate the development of human erythroid/myeloid progenitor cells and their progeny in normal differentiation and in the chimeric state of transfusion and allogeneic HSCT. Furthermore, novel technology platforms for optimal HSCT graft engineering in the setting of recipient-donor tissue antigen disparity were investigated. We utilized two in vitro culture systems (Paper II) to obtain cells of advanced neutrophil and erythroid maturation from adult human CD34+ bone marrow cells, and to study the relationship between clonogenicity, gene expression and phenotype during early myeloid development. Cells were sorted by fluorescence-activated-cell-sorting (FACS) according to selected surface markers into defined populations of sequential developmental stages for further analysis. Thus the expression profiles of several genes could to be directly correlated to changes in clonogenic potential and to lineage commitment, as defined by surface expression of CD15 (neutrophil differentiation) and group A antigen (eryhtroid differentiation). With regard to the established surface markers for neutrofil and erythroid lineage-affiliation, we investigated (Paper I) the origin of reappearing recipient-derived RBC in a patient with relapsed myelodysplastic syndrome (MDS) following allogeneic HSCT. The presence of the original cytogenetic 20q-deletion in myeloid cell populations of different maturity, suggested an origin common to the original myeloid malignant clone and the reappearing autologous RBC, i.e. a progenitor cell with preserved granulocyte/monocyte and megakaryocyte/erythroid potential. We continued our study of chimeric post-HSCT recipients by addressing (paper V) the phenomenon of blood group A/B antigen acquisition by donor group O RBC, following transfusion or ABO-incompatible HSCT. Using a highly sensitive flow cytometry assay, donor group O RBC were found to express variable levels of acquired antigen, ranging from very small amounts in non-secretor individuals to ABO-subgroup levels in group A1 secretors. Our findings support the major role of A/B antigen adsorption from secretor plasma, but indicate a secretor-independent mechanism for A/B-antigen acquisition. To improve graft processing in HLA-mismatched HSCT, we investigated (Paper III) and optimized the performance of a novel magnetic cell sorting program (Depletion 3.1, CliniMACS System), for large-scale direct depletion of T-cells from peripheral blood progenitor cells (PBPC). The optimized D3.1 program can be utilized for large-scale, time saving direct T-cell depletion with excellent recovery of CD34+ cells and an effective reduction of T-cell numbers. Furthermore, a novel micro-chip based acoustophoresis technique was invesigated (paper IV) for removal of platelets from PBPC products and was found to efficiently deplete PBPC samples of intact platelets, whilst preserving the target leukocyte fraction, cell viability and progenitor cell colony-forming ability. Acoustophoresis is, thus, an interesting technology to improve current cell processing methods. In summary, this work has provided knowledge on differentiation-associated changes in human myeloid development. Surface restriction markers for erythroid (group A antigen) and neutrophil differentiation (CD15) have been established and utilized to investigate the origin of reappearing host cells post-HSCT. In a clinical context, the level of A/B-antigen found to be acquired by donor group O RBC following transfusion or HSCT suggests implications for plasma component selection. Moreover, current clinical cell separation methodology was improved and the novel acoustophoresis technology was demonstrated to provide efficient platelet depletion, with a potential application in future clinical graft engineering

Clonogenicity, gene expression and phenotype during neutrophil versus erythroid differentiation of cytokine-stimulated CD34 human marrow cells in vitro.

With the objective to correlate clonogenicity, gene expression and phenotype during differentiation, human bone marrow CD34+ cells were cultured in vitro to stimulate erythroid or neutrophil development, and sorted into five subpopulations according to their surface expression of CD15/CD33 and blood group antigen A/CD117 respectively. Sorted cells were cultured in methylcellulose and analysed by real-time reverse transcription polymerase chain reaction for expression of neutrophil and erythroid marker genes. Surface expression of CD15 coincided with restriction to neutrophil/monocyte differentiation and A antigen with restriction to erythroid differentiation. GATA-2 mRNA was down-regulated during both neutrophil and erythroid maturation, whereas GATA-1, SCL, ABO, erythropoietin receptor, Kell, glycophorin A, β-globin and α-haemoglobin stabilizing protein were up-regulated during erythroid differentiation and silenced during neutrophil differentiation. CCAAT/enhancer-binding protein (C/EBP)-α, PU.1, granulocyte colony-stimulating factor receptor, PR3, C/EBP-e and lactoferrin were sequentially expressed during neutrophil differentiation but rapidly down-regulated during the early erythroid stages. Nuclear factor erythroid-derived 2 (NF-E2) and glycophorin C were expressed both during neutrophil and erythroid differentiation. Our data support the notion of early expression of several lineage-associated genes prior to actual lineage commitment, defined by surface expression of CD15 and A antigen as markers for definitive neutrophil/monocyte and erythroid differentiation respectively. Previous findings, primarily from cell lines and mouse models, have been extended to adult human haematopoiesis

Efficient Removal of Platelets from Peripheral Blood Progenitor Cell Products Using a Novel Micro-Chip Based Acoustophoretic Platform

Excessive collection of platelets is an unwanted side effect in current centrifugation-based peripheral blood progenitor cell (PBPC) apheresis. We investigated a novel microchip-based acoustophoresis technique, utilizing ultrasonic standing wave forces for the removal of platelets from PBPC products. By applying an acoustic standing wave field onto a continuously flowing cell suspension in a micro channel, cells can be separated from the surrounding media depending on their physical properties

Novel treatment of severe combined immunodeficiency utilizing ex-vivo T-cell depleted haploidentical hematopoietic stem cell transplantation and CD45RA+ depleted donor lymphocyte infusions.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment available for severe combined immunodeficiency (SCID); although, there is a high incidence of severe infections and an increased risk of graft-versus host-disease (GvHD) with HSCT. Early intervention is a crucial prognostic factor and a HLA-haploidentical parental donor is often available. Haploidentical HSCT protocols utilizing extensively ex vivo T-cell depleted grafts (CliniMACs system) have proven efficient in preventing GvHD, but cause a delay in early T-cell recovery that increases the risk of viral infections. Here, we present a novel approach for treating SCID that combines selective depletion of GvHD-inducing alpha/beta (α/β) T-cells from the haploidentical HSCT graft with a subsequent donor lymphocyte infusion (DLI) enriched for CD45RO+ memory T-cells

Affinity-Bead-Mediated Enrichment of CD8+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Acoustophoresis

Acoustophoresis is a technique that applies ultrasonic standing wave forces in a microchannel to sort cells depending on their physical properties in relation to the surrounding media. Cell handling and separation for research and clinical applications aims to efficiently separate specific cell populations. Here, we investigated the sorting of CD8 lymphocytes from peripheral blood progenitor cell (PBPC) products by affinity-bead-mediated acoustophoresis. PBPC samples were obtained from healthy donors (n = 4) and patients (n = 18). Mononuclear cells were labeled with anti-CD8-coated magnetic beads and sorted on an acoustophoretic microfluidic device and by standard magnetic cell sorting as a reference method. CD8 lymphocytes were acoustically sorted with a mean purity of 91% ± 8% and a median separation efficiency of 63% (range 15.1%–90.5%) as compared to magnetic sorting (purity 91% ± 14%, recovery 29% (range 5.1%–47.3%)). The viability as well as the proliferation capacity of sorted lymphocytes in the target fraction were unimpaired and, furthermore, hematopoietic progenitor cell assay revealed a preserved clonogenic capacity post-sorting. Bead-mediated acoustophoresis can, therefore, be utilized to efficiently sort less frequent CD8+ lymphocytes from PBPC products in a continuous flow mode while maintaining cell viability and functional capacity of both target and non-target fractions

Label-free neuroblastoma cell separation from hematopoietic progenitor cell products using acoustophoresis - towards cell processing of complex biological samples

Processing of complex cell preparations such as blood and peripheral blood progenitor cell (PBPC) transplants using label-free technologies is challenging. Transplant-contaminating neuroblastoma cells (NBCs) can contribute to relapse, and we therefore aimed to provide proof-of-principle evidence that label-free acoustophoretic separation can be applied for diagnostic NBC enrichment and removal ("purging") from human blood and PBPC products. Neuroblastoma cells spiked into blood and PBPC preparations served as model systems. Acoustophoresis enabled to enrich NBCs from mononuclear peripheral blood cells and PBPC samples with recovery rates of up to 60-97%. When aiming at high purity, NBC purities of up to 90% were realized, however, compromising recovery. Acoustophoretic purging of PBPC products allowed substantial tumour cell depletion of 1.5-2.3 log. PBPC loss under these conditions was considerable (>43%) but could be decreased to less than 10% while still achieving NBC depletion rates of 60-80%. Proliferation of cells was not affected by acoustic separation. These results provide first evidence that NBCs can be acoustically separated from blood and stem cell preparations with high recovery and purity, thus indicating that acoustophoresis is a promising technology for the development of future label-free, non-contact cell processing of complex cell products