Expansion of primitive human hematopoietic stem cells by culture in a zwitterionic hydrogel.

The ability to expand hematopoietic stem and progenitor cells (HSPCs) ex vivo is critical to fully realize the potential of HSPC-based therapies. In particular, the application of clinically effective therapies, such as cord blood transplantation, has been impeded because of limited HSPC availability. Here, using 3D culture of human HSPCs in a degradable zwitterionic hydrogel, we achieved substantial expansion of phenotypically primitive CD34+ cord blood and bone-marrow-derived HSPCs. This culture system led to a 73-fold increase in long-term hematopoietic stem cell (LT-HSC) frequency, as demonstrated by limiting dilution assays, and the expanded HSPCs were capable of hematopoietic reconstitution for at least 24 weeks in immunocompromised mice. Both the zwitterionic characteristics of the hydrogel and the 3D format were important for HSPC self-renewal. Mechanistically, the impact of 3D zwitterionic hydrogel culture on mitigating HSPC differentiation and promoting self-renewal might result from an inhibition of excessive reactive oxygen species (ROS) production via suppression of O2-related metabolism. HSPC expansion using zwitterionic hydrogels has the potential to facilitate the clinical application of hematopoietic-stem-cell therapies.

TALEN-Mediated Gene Editing of HBG in Human Hematopoietic Stem Cells Leads to Therapeutic Fetal Hemoglobin Induction.

Elements within the γ-hemoglobin promoters (HBG1 and HBG2) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the HBG1 promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous HBG promoters to de-repress fetal hemoglobin. Transfection of human CD34+ cells with TALEN mRNA resulted in indel generation in HBG1 (43%) and HBG2 (74%) including the 13-bp HPFH deletion (∼6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in γ-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34+ cells in vivo in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human γ-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the γ-hemoglobin promoter leads to high levels of fetal hemoglobin expression in vitro and in vivo, suggesting that this approach can provide therapeutic benefit in patients with SCD or ß-thalassemia.

SIRT1 is dispensable for function of hematopoietic stem cells in adult mice.

SIRT1 is an NAD(+)-dependent histone deacetylase implicated in the establishment of the primitive hematopoietic system during mouse embryonic development. However, investigation of the role of SIRT1 in adult hematopoiesis has been complicated by the high perinatal mortality of SIRT1-deficient mice (SIRT1(-/-)). We performed a comprehensive in vivo study of the hematopoietic stem cell (HSC) compartment in adult SIRT1(-/-) mice and show that, apart from anemia and leukocytosis in older mice, the production of mature blood cells, lineage distribution within hematopoietic organs, and frequencies of the most primitive HSC populations are comparable to those of wild-type littermate controls. Furthermore, we show that SIRT1-deficient BM cells confer stable long-term reconstitution in competitive repopulation and serial transplantation experiments. The results of the present study rule out an essential physiologic role for cell-autonomous SIRT1 signaling in the maintenance of the adult HSC compartment in mice.

Anatomical compartments modify the response of human hematopoietic cells to a mitogenic signal.

Methods for specifically regulating transplanted cells have many applications in gene and cell therapy. We examined the response of human cord blood CD34+ cells to a specific mitotic signal in vivo. Using a conditional signaling molecule (F36VMpl) that is specifically activated by an artificial ligand called a chemical inducer of dimerization (CID), human hematopoietic cells transplanted into immune deficient mice were induced to proliferate. Only differentiating erythroid precursors and multipotential and erythroid progenitors (colony-forming unit [CFU]-mix and burst forming unitserythroid [BFUe]) responded; however, the nature of the response differed markedly between bone marrow and spleen. In the marrow, F36VMpl induced a 12- to 17-fold expansion of differentiated erythroid precursors and a loss of CFU-mix and BFUe. In the spleen, F36VMpl induced a marked rise in BFUe and CFU-mix and, relative to marrow, a much less prominent rise in more mature red cells. Clonal analysis was most consistent with the interpretation that the spleen and bone marrow differentially regulate the response of human progenitors to a mitotic signal, possibly influencing progenitor expansion versus differentiation. These findings establish CIDs as in vivo growth factors for human hematopoietic cells.

Conditionally cleavable radioimmunoconjugates: a novel approach for the release of radioisotopes from radioimmunoconjugates.

One of the limitations of therapy with radiolabeled monoclonal antibodies (mAbs) is that significant toxicities can arise from circulating non-tumor-bound radiolabeled conjugate. Here, we describe a new method to reduce systemic radiation exposure from radiolabeled mAbs involving the attachment of the radioisotope through a linker that can be cleaved by an administered enzyme. To demonstrate the feasibility of this approach, we prepared a conditionally cleavable radioimmunoconjugate (RIC) composed of (131)I-labeled cephalosporin conjugated to Tositumomab, a mAb against the CD20 antigen. The cleavable RIC bound antigen identically to directly iodinated antibody, and in the presence of beta-lactamase, about 80-85% of the radioisotope was released. In vivo studies in mice revealed that the cleavable RIC and the directly iodinated anti-CD20 antibody had similar biodistribution patterns. Systemically administered beta-lactamase induced a 2-3-fold decrease in the percent injected dose (ID) of the cleavable RIC/g of blood, marrow, spleen, lung, and liver 1 h after enzyme treatment, and a 4-6-fold decrease 20 h after enzyme treatment. This was accompanied by a 20-fold increase in % ID/g in urine 1 h after enzyme treatment, indicating that the released radiolabel was rapidly excreted through the kidneys. In mice with human tumor xenografts, there was no decrease in the %ID/g in tumor 1 h after enzyme treatment, but by 4 h after enzyme injection, decreases in tumor radioactive content began to diminish the targeting advantage. These studies demonstrate that the cleavable RIC substrate is able to bind to tumor antigens and localize within human tumor xenografts and that accelerated systemic clearance can be induced with beta-lactamase.