Ingbritt Åstrand-Grundström

Upregulation of Flt3 Expression within the Bone Marrow Lin−Sca1+c-kit+ Stem Cell Compartment Is Accompanied by Loss of Self-Renewal Capacity

Flt3 has emerged as a potential regulator of hematopoietic stem cells (HSC). Sixty percent of cells in the mouse marrow Lin(-)Sca1(+)c-kit(+) HSC pool expressed flt3. Although single cell cloning showed comparable high proliferative, myeloid, B, and T cell potentials of Lin(-)Sca1(+)c-kit(+)flt3(+) and Lin(-)Sca1(+)c-kit(+)flt3(-) cells, only Lin(-)Sca1(+)c-kit(+)flt3(-) cells supported sustained multilineage reconstitution. In striking contrast, Lin(-)Sca1(+)c-kit(+)flt3(+) cells rapidly and efficiently reconstituted B and T lymphopoiesis, whereas myeloid reconstitution was exclusively short term. Unlike c-kit, activation of flt3 failed to support survival of HSC, whereas only flt3 mediated survival of Lin(-)Sca1(+)c-kit(+)flt3(+) reconstituting cells. Phenotypic and functional analysis support that Lin(-)Sca1(+)c-kit(+)flt3(+) cells are progenitors for the common lymphoid progenitor. Thus, upregulation of flt3 expression on Lin(-)Sca1(+)c-kit(+) HSC cells is accompanied by loss of self-renewal capacity but sustained lymphoid-restricted reconstitution potential.

Distinct patterns of hematopoietic stem cell involvement in acute lymphoblastic leukemia

The cellular targets of primary mutations and malignant transformation remain elusive in most cancers. Here, we show that clinically and genetically different subtypes of acute lymphoblastic leukemia (ALL) originate and transform at distinct stages of hematopoietic development. Primary ETV6-RUNX1 (also known as TEL-AML1) fusions and subsequent leukemic transformations were targeted to committed B-cell progenitors. Major breakpoint BCR-ABL1 fusions (encoding P210 BCR-ABL1) originated in hematopoietic stem cells (HSCs), whereas minor BCR-ABL1 fusions (encoding P190 BCR-ABL1) had a B-cell progenitor origin, suggesting that P190 and P210 BCR-ABL1 ALLs represent largely distinct tumor biological and clinical entities. The transformed leukemia-initiating stem cells in both P190 and P210 BCR-ABL1 ALLs had, as in ETV6-RUNX1 ALLs, a committed B progenitor phenotype. In all patients, normal and leukemic repopulating stem cells could successfully be separated prospectively, and notably, the size of the normal HSC compartment in ETV6-RUNX1 and P190 BCR-ABL1 ALLs was found to be unaffected by the expansive leukemic stem cell population.

Persistent malignant stem cells in del(5q) myelodysplasia in remission.

BACKGROUND The in vivo clinical significance of malignant stem cells remains unclear. METHODS Patients who have the 5q deletion (del[5q]) myelodysplastic syndrome (interstitial deletions involving the long arm of chromosome 5) have complete clinical and cytogenetic remissions in response to lenalidomide treatment, but they often have relapse. To determine whether the persistence of rare but distinct malignant stem cells accounts for such relapses, we examined bone marrow specimens obtained from seven patients with the del(5q) myelodysplastic syndrome who became transfusion-independent while receiving lenalidomide treatment and entered cytogenetic remission. RESULTS Virtually all CD34+, CD38+ progenitor cells and stem cells that were positive for CD34 and CD90, with undetectable or low CD38 (CD38−/low), had the 5q deletion before treatment. Although lenalidomide efficiently reduced these progenitors in patients in complete remission, a larger fraction of the minor, quiescent, CD34+,CD38-/low, CD90+ del(5q) stem cells as well as functionally defined del(5q) stem cells remained distinctly resistant to lenalidomide. Over time, lenalidomide resistance developed in most of the patients in partial and complete remission, with recurrence or expansion of the del(5q) clone and clinical and cytogenetic progression. CONCLUSIONS In these patients with the del(5q) myelodysplastic syndrome, we identified rare and phenotypically distinct del(5q) myelodysplastic syndrome stem cells that were also selectively resistant to therapeutic targeting at the time of complete clinical and cytogenetic remission. (Funded by the EuroCancerStemCell Consortium and others.)

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

Background 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. Study Design and Methods PBPC samples were obtained from patients (n = 15) and healthy donors (n = 6) and sorted on an acoustophoresis-chip. The acoustic force was set to separate leukocytes from platelets into a target fraction and a waste fraction, respectively. The PBPC samples, the target and the waste fractions were analysed for cell recovery, purity and functionality. Results The median separation efficiency of leukocytes to the target fraction was 98% whereas platelets were effectively depleted by 89%. PBPC samples and corresponding target fractions were similar in the percentage of CD34+ hematopoetic progenitor/stem cells as well as leukocyte/lymphocyte subset distributions. Median viability was 98%, 98% and 97% in the PBPC samples, the target and the waste fractions, respectively. Results from hematopoietic progenitor cell assays indicated a preserved colony-forming ability post-sorting. Evaluation of platelet activation by P-selectin (CD62P) expression revealed a significant increase of CD62P+ platelets in the target (19%) and waste fractions (20%), respectively, compared to the PBPC input samples (9%). However, activation was lower when compared to stored blood bank platelet concentrates (48%). Conclusion Acoustophoresis can be utilized to efficiently deplete PBPC samples of platelets, whilst preserving the target stem/progenitor cell and leukocyte cell populations, cell viability and progenitor cell colony-forming ability. Acoustophoresis is, thus, an interesting technology to improve current cell processing methods.

MDS/AML-associated cytogenetic abnormalities in multiple myeloma and monoclonal gammopathy of undetermined significance: Evidence for frequent de novo occurrence and multipotent stem cell involvement of del(20q)

Multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are characterized cytogenetically by 14q32 rearrangements, −13/13q−, and various trisomies. Occasionally, karyotypic patterns characteristic of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) occur in MM, often signifying therapy‐related (t)‐MDS/t‐AML. Comparison of cytogenetic features in all published MMs (n = 993) and t‐MDS/t‐AML post‐MM (n = 117) revealed significant differences in complexity and ploidy levels and in most genomic changes. Thus, these features often can be used to distinguish between MM and t‐MDS/t‐AML. Rarely, myeloid‐associated aberrations are detected in MM without any signs of MDS/AML. To characterize such abnormalities in MM/MGUS, we ascertained all 122 MM and 26 MGUS/smoldering MM (SMM) cases analyzed in our department. Sixty‐six (54%) MMs and 8 (31%) MGUS/SMMs were karyotypically abnormal, of which 6 (9%) MMs and 3 (38%) MGUS/SMMs displayed myeloid abnormalities, that is, +8 (1 case) and 20q− (8 cases) as the sole anomalies, without any evidence of MDS/AML. One patient developed AML, whereas no MDS/AML occurred in the remaining 8 patients. In one MGUS with del(20q), fluorescence in situ hybridization analyses revealed its presence in CD34+CD38− (hematopoietic stem cells), CD34+CD38+ (progenitors), CD19+ (B cells), and CD15+ (myeloid cells). The present data indicate that 20q− occurs in 10% of karyotypically abnormal MM/MGUS cases and that it might arise at a multipotent progenitor/stem cell level.

Efficient Purification of CD4+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Affinity Bead Acoustophoresis

Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively isolate or deplete specific cell populations, utilizing primarily magnetic or fluorescence activated sorting methods. Here, we investigated the performance of microfluidic acoustophoresis for the separation of lymphocyte subsets from PBPC, and present a novel method for affinity‐bead‐mediated acoustic separation of cells which can otherwise not be acoustically discriminated. As the acoustic force on a particle depends on particle size, density and compressibility, targeting of cells by affinity specific beads will generate cell‐bead complexes that exhibit distinct acoustic properties relative to nontargeted cells and are, thus, possible to isolate. To demonstrate this, PBPC samples (n = 22) were obtained from patients and healthy donors. Following density gradient centrifugation, cells were labeled with anti‐CD4‐coated magnetic beads (Dynal) and isolated by acoustophoresis and, for comparison, standard magnetic cell sorting technique in parallel. Targeted CD4+ lymphocytes were acoustically isolated with a mean (±SD) purity of 87 ± 12%, compared with 96 ± 3% for control magnetic sorting. Viability of sorted cells was 95 ± 4% (acoustic) and 97 ± 3% (magnetic), respectively. The mean acoustic separation efficiency of CD4+ lymphocytes to the target fraction was 65 ± 22%, compared with a mean CD4+ lymphocyte recovery of 56 ± 15% for magnetic sorting. Functional testing of targeted CD4+ lymphocytes demonstrated unimpaired mitogen‐mediated proliferation capacity and cytokine production. Hematopoietic progenitor cell assays revealed a preserved colony forming ability of nontarget cells post sorting. We conclude that the acoustophoresis platform can be utilized to efficiently isolate bead‐labeled CD4+ lymphocytes from PBPC samples in a continuous flow format, with preserved functional capacity of both target and nontarget cells. These results open up for simultaneous affinity‐bead‐mediated separation of multiple cell populations, something which is not possible with current standard magnetic cell separation technology.

Expression of Integrin α2 Receptor in Human Cord Blood CD34+CD38−CD90+ Stem Cells Engrafting Long-Term in NOD/SCID-IL2Rγcnull Mice†‡

Human hematopoietic stem cells reside in the CD34+CD38−CD90+ population in cord blood and bone marrow. However, this cell fraction is heterogeneous, and the phenotype of the rare primitive stem cells remains poorly defined. We here report that primitive cord blood CD34+CD38−CD90+ stem cells, with the ability to reconstitute NOD/SCID‐IL2Rγcnull (NSG) mice long‐term, at 24 weeks after transplantation, can be prospectively isolated at an increased purity by using integrin α2 receptor as an additional stem cell marker. Using a limiting dilution transplantation assay, we found a highly significant enrichment of multilineage reconstituting stem cells in the CD34+CD38−CD90+ cell fraction expressing the integrin α2 receptor, with a frequency of 1/29 cells, as compared to a frequency of 1/157 in the corresponding integrin α2− cells. In line with this, long‐term reconstituting stem cells within the cord blood CD34+CD38− cell population were significantly enriched in the integrin α2+ fraction, while stem cells and progenitors reconstituting short‐term, at 8–12 weeks, were heterogeneous in integrin α2 expression. Global gene expression profiling revealed that the lineage‐marker negative (Lin−) CD34+CD38−CD90+CD45RA− integrin α2+ cell population was molecularly distinct from the integrin α2− cell population and the more mature Lin−CD34+CD38−CD90−CD45RA− cell population. Our findings identify integrin α2 as a novel stem cell marker, which improves prospective isolation of the primitive human hematopoietic stem cells within the CD34+CD38−CD90+ cell population for experimental and therapeutic stem cell applications. STEM CELLS2013;31:360–371