Stefan Radtke

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect the Fetal Brain After Hypoxia-Ischemia

Preterm neonates are susceptible to perinatal hypoxic‐ischemic brain injury, for which no treatment is available. In a preclinical animal model of hypoxic‐ischemic brain injury in ovine fetuses, we have demonstrated the neuroprotective potential of systemically administered mesenchymal stromal cells (MSCs). The mechanism of MSC treatment is unclear but suggested to be paracrine, through secretion of extracellular vesicles (EVs). Therefore, we investigated in this study the protective effects of mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs) in a preclinical model of preterm hypoxic‐ischemic brain injury. Ovine fetuses were subjected to global hypoxia‐ischemia by transient umbilical cord occlusion, followed by in utero intravenous administration of MSC‐EVs. The therapeutic effects of MSC‐EV administration were assessed by analysis of electrophysiological parameters and histology of the brain. Systemic administration of MSC‐EVs improved brain function by reducing the total number and duration of seizures, and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC‐EV treatment. Our data demonstrate that MSC‐EV treatment might provide a novel strategy to reduce the neurological sequelae following hypoxic‐ischemic injury of the preterm brain. Our study results suggest that a cell‐free preparation comprising neuroprotective MSC‐EVs could substitute MSCs in the treatment of preterm neonates with hypoxic‐ischemic brain injury, thereby circumventing the potential risks of systemic administration of living cells.

CD133 allows elaborated discrimination and quantification of haematopoietic progenitor subsets in human haematopoietic stem cell transplants

The success of haematopoietic stem cell (HSC) transplantation largely depends on numbers of transplanted HSCs, which reside in the CD34+ populations of bone marrow (BM), peripheral blood stem cells (PBSC) and umbilical cord blood (UCB). More specifically HSCs reside in the CD38low/− subpopulation, which cannot be objectively discriminated from mature CD34+ CD38+ progenitors. Thus, better marker combinations for the quantification of more primitive haematopoietic stem and progenitor cells in transplants are required. Recently, by combining CD34 and CD133 we could clearly distinguish CD133+ CD34+ multipotent and lympho‐myeloid from CD133low CD34+ erythro‐myeloid progenitors in UCB samples. To qualify the assessment of CD133 for routine quality control of adult HSC sources, we analysed the developmental potentials of CD133+ and CD133low subpopulations in BM and PBSC. Similar to UCB, CD133 expression objectively discriminated functionally distinct subpopulations in adult HSC sources. By implementing anti‐CD45RA staining, which separates multipotent (CD133+ CD34+ CD45RA−) from lympho‐myeloid (CD133+ CD34+ CD45RA+) progenitor fractions, UCB was found to contain 2–3 times higher multipotent progenitor frequencies than BM and PBSC. To test for the consistency of CD133 expression, we compared CD133+ CD34+ contents of 128 UCB samples with maternal and obstetrical factors and obtained similar correlations to related studies focusing on CD34+ cell contents. In conclusion, implementation of anti‐CD133 staining into existing routine panels will improve the quality control analyses for HSC transplants.

Suppression of luteinizing hormone enhances HSC recovery after hematopoietic injury

There is a substantial unmet clinical need for new strategies to protect the hematopoietic stem cell (HSC) pool and regenerate hematopoiesis after radiation injury from either cancer therapy or accidental exposure. Increasing evidence suggests that sex hormones, beyond their role in promoting sexual dimorphism, regulate HSC self-renewal, differentiation, and proliferation. We and others have previously reported that sex-steroid ablation promotes bone marrow (BM) lymphopoiesis and HSC recovery in aged and immunodepleted mice. Here we found that a luteinizing hormone (LH)-releasing hormone antagonist (LHRH-Ant), currently in wide clinical use for sex-steroid inhibition, promoted hematopoietic recovery and mouse survival when administered 24 h after an otherwise-lethal dose of total-body irradiation (L-TBI). Unexpectedly, this protective effect was independent of sex steroids and instead relied on suppression of LH levels. Human and mouse long-term self-renewing HSCs (LT-HSCs) expressed high levels of the LH/choriogonadotropin receptor (LHCGR) and expanded ex vivo when stimulated with LH. In contrast, the suppression of LH after L-TBI inhibited entry of HSCs into the cell cycle, thus promoting HSC quiescence and protecting the cells from exhaustion. These findings reveal a role of LH in regulating HSC function and offer a new therapeutic approach for hematopoietic regeneration after hematopoietic injury.

A Nonhuman Primate Transplantation Model to Evaluate Hematopoietic Stem Cell Gene Editing Strategies for β-Hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is a promising approach for the treatment of β-hemoglobinopathies and the targeting of genes involved in HbF regulation is under intensive investigation. Here, we established a nonhuman primate (NHP) transplantation model to evaluate hematopoietic stem cell (HSC)-based gene editing strategies aimed at reactivating HbF. We first characterized the transient HbF induction to autologous HSC transplantation in pigtailed macaques, which was comparable in duration and amplitude to that of human patients. After validating function of the HbF repressor BCL11A in NHPs, we transplanted a pigtailed macaque with CD34+ cells electroporated with TALE nuclease mRNA targeting the BCL11A coding sequence. In vivo gene editing levels were low, but some BCL11A deletions were detected as late as 200 days post-transplantation. HbF production, as determined by F-cell staining and γ-globin expression, was slightly increased in this animal as compared to transplant controls. We also provided proof-of-concept results for the selection of edited NHP CD34+ cells in culture following integration of the P140K/MGMT cassette at the BCL11A locus. In summary, the NHP model described here will allow the testing of novel therapeutic approaches for hemoglobinopathies and should facilitate clinical translation.

A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates

A population of hematopoietic stem cells with superior engraftment and repopulating abilities has been identified in nonhuman primates. Refining the gold standard CD34-positive hematopoietic cells are the gold standard for stem cell therapy and transplantation of stem cell–enriched grafts. However, most of the cells within this population will not contribute to engraftment. Using a robust nonhuman primate transplantation model, Radtke et al. identified a stem cell–enriched subpopulation of CD34-positive cells that was exclusively responsible for multilineage engraftment. The cell dose of this subpopulation correlated with neutrophil and platelet recovery and reliably predicted overall transplant success. The authors observed phenotypic and transcriptomic similarities between these cells and human hematopoietic cells with high engraftment and repopulating potential. These data suggest a refined subpopulation of CD34-positive cells for use in transplantation and gene therapy/editing approaches. Hematopoietic reconstitution after bone marrow transplantation is thought to be driven by committed multipotent progenitor cells followed by long-term engrafting hematopoietic stem cells (HSCs). We observed a population of early-engrafting cells displaying HSC-like behavior, which persisted long-term in vivo in an autologous myeloablative transplant model in nonhuman primates. To identify this population, we characterized the phenotype and function of defined nonhuman primate hematopoietic stem and progenitor cell (HSPC) subsets and compared these to human HSPCs. We demonstrated that the CD34+CD45RA−CD90+ cell phenotype is highly enriched for HSCs. This population fully supported rapid short-term recovery and robust multilineage hematopoiesis in the nonhuman primate transplant model and quantitatively predicted transplant success and time to neutrophil and platelet recovery. Application of this cell population has potential in the setting of HSC transplantation and gene therapy/editing approaches.

The frequency of multipotent CD133(+)CD45RA(-)CD34(+) hematopoietic stem cells is not increased in fetal liver compared with adult stem cell sources.

The cell surface marker CD133 has been used to describe a revised model of adult human hematopoiesis, with hematopoietic stem cells and multipotent progenitors (HSCs/MPPs: CD133(+)CD45RA(-)CD34(+)) giving rise to lymphomyeloid-primed progenitors (LMPPs: CD133(+)CD45RA(+)CD34(+)) and erythromyeloid progenitors (EMPs: CD133(low)CD45RA(-)CD34(+)). Because adult and fetal hematopoietic stem and progenitor cells (HSPCs) differ in their gene expression profile, differentiation capabilities, and cell surface marker expression, we were interested in whether the reported segregation of lineage potentials in adult human hematopoiesis would also apply to human fetal liver. CD133 expression was easily detected in human fetal liver cells, and the defined hematopoietic subpopulations were similar to those found for adult HSPCs. Fetal HSPCs were enriched for EMPs and HSCs/MPPs, which were primed toward erythromyeloid differentiation. However, the frequency of multipotent CD133(+)CD45RA(-)CD34(+) HSPCs was much lower than previously reported and comparable to that of umbilical cord blood. We noted that engraftment in NSG (NOD scid gamma [NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ])xa0mice was driven mostly by LMPPs, confirming recent findings that repopulation in mice is not a unique feature of multipotent HSCs/MPPs. Thus, our data challenge the general assumption that human fetal liver contains a greater percentage of multipotent HSCs/MPPs than any adult HSC source, and the mouse model may have to be re-evaluated with respect to the type of readout it provides.

Human mesenchymal and murine stromal cells support human lympho-myeloid progenitor expansion but not maintenance of multipotent haematopoietic stem and progenitor cells

ABSTRACT A major goal in haematopoietic stem cell (HSC) research is to define conditions for the expansion of HSCs or multipotent progenitor cells (MPPs). Since human HSCs/MPPs cannot be isolated, NOD/SCID repopulating cell (SRC) assays emerged as the standard for the quantification of very primitive haematopoietic cell. However, in addition to HSCs/MPPs, lympho-myeloid primed progenitors (LMPPs) were recently found to contain SRC activities, challenging this assay as clear HSC/MPP readout. Because our revised model of human haematopoiesis predicts that HSCs/MPPs can be identified as CD133+CD34+ cells containing erythroid potentials, we investigated the potential of human mesenchymal and conventional murine stromal cells to support expansion of HSCs/MPPs. Even though all stromal cells supported expansion of CD133+CD34+ progenitors with long-term myeloid and long-term lymphoid potentials, erythroid potentials were exclusively found within erythro-myeloid CD133lowCD34+ cell fractions. Thus, our data demonstrate that against the prevailing assumption co-cultures on human mesenchymal and murine stromal cells neither promote expansion nor maintenance of HSCs and MPPs.

Corrigendum: Suppression of luteinizing hormone enhances HSC recovery after hematopoietic injury

This corrects the article DOI: 10.1038/nm.4470.