Mattias Magnusson

Development of an adenoviral vector system with adenovirus serotype 35 tropism; efficient transient gene transfer into primary malignant hematopoietic cells.

A paucity of coxsackie adenovirus receptor (CAR) hampers the adenovirus serotype 5 (Ad5)‐based vector‐mediated gene transfer into malignant hematopoietic cells. Fiber‐retargeted adenoviral vectors with species B tropism can potentially bypass the CAR requirement and facilitate efficient gene transfer into malignant hematopoietic cells.

Reduced proliferative capacity of hematopoietic stem cells deficient in Hoxb3 and Hoxb4

ABSTRACT Several homeobox transcription factors, such as HOXB3 and HOXB4, have been implicated in regulation of hematopoiesis. In support of this, studies show that overexpression of HOXB4 strongly enhances hematopoietic stem cell regeneration. Here we find that mice deficient in both Hoxb3 and Hoxb4 have defects in endogenous hematopoiesis with reduced cellularity in hematopoietic organs and diminished number of hematopoietic progenitors without perturbing lineage commitment. Analysis of embryonic day 14.5 fetal livers revealed a significant reduction in the hematopoietic stem cell pool, suggesting that the reduction in cellularity observed postnatally is due to insufficient expansion during fetal development. Primitive Lin− ScaI+ c-kit+ hematopoietic progenitors lacking Hoxb3 and Hoxb4 displayed impaired proliferative capacity in vitro. Similarly, in vivo repopulating studies of Hoxb3/Hoxb4-deficient hematopoietic cells resulted in lower repopulating capability compared to normal littermates. Since no defects in homing were observed, these results suggest a slower regeneration of mutant HSC. Furthermore, treatment with cytostatic drugs demonstrated slower cell cycle kinetics of hematopoietic stem cells deficient in Hoxb3 and Hoxb4, resulting in increased tolerance to antimitotic drugs. Collectively, these data suggest a direct physiological role of Hoxb4 and Hoxb3 in regulating stem cell regeneration and that these genes are required for maximal proliferative response.

The first trimester human placenta is a site for terminal maturation of primitive erythroid cells

Embryonic hematopoiesis starts via the generation of primitive red blood cells (RBCs) that satisfy the embryos immediate oxygen needs. Although primitive RBCs were thought to retain their nuclei, recent studies have shown that primitive RBCs in mice enucleate in the fetal liver. It has been unknown whether human primitive RBCs enucleate, and what hematopoietic site might support this process. Our data indicate that the terminal maturation and enucleation of human primitive RBCs occurs in first trimester placental villi. Extravascular ζ-globin(+) primitive erythroid cells were found in placental villi between 5-7 weeks of development, at which time the frequency of enucleated RBCs was higher in the villous stroma than in circulation. RBC enucleation was further evidenced by the presence of primitive reticulocytes and pyrenocytes (ejected RBC nuclei) in the placenta. Extravascular RBCs were found to associate with placental macrophages, which contained ingested nuclei. Clonogenic macrophage progenitors of fetal origin were present in the chorionic plate of the placenta before the onset of fetoplacental circulation, after which macrophages had migrated to the villi. These findings indicate that placental macrophages may assist the enucleation process of primitive RBCs in placental villi, implying an unexpectedly broad role for the placenta in embryonic hematopoiesis.

Transient disruption of autocrine TGF-beta signaling leads to enhanced survival and proliferation potential in single primitive human hemopoietic progenitor cells.

Hemopoietic stem cells (HSCs) are maintained at relative quiescence by the balance between the positive and negative regulatory factors that stimulate or inhibit their proliferation. Blocking the action of negative regulatory factors may provide a new approach for inducing HSCs into proliferation. A variety of studies have suggested that TGF-β negatively regulates cell cycle progression of HSCs. In this study, a dominant negatively acting mutant of TGF-β type II receptor (TβRIIDN) was transiently expressed in HSCs by using adenoviral vector-mediated gene delivery, such that the effects of disrupting the autocrine TGF-β signaling in HSCs can be directly examined at a single cell level. Adenoviral vectors allowing the expression of TβRIIDN and green fluorescence protein in the same CD34+CD38−Lin− cells were constructed. Overexpression of TβRIIDN specifically disrupted TGF-β-mediated signaling. Autocrine TGF-β signaling in CD34+CD38−Lin− cells was studied in single cell assays under serum-free conditions. Transient blockage of autocrine TGF-β signaling in CD34+CD38−Lin− cells enhanced their survival. Furthermore, the overall proliferation potential and proliferation kinetics in these cells were significantly enhanced compared with the CD34+CD38−Lin− cells expressing green fluorescence protein alone. Therefore, we have successfully blocked the autocrine TGF-β-negative regulatory loop of primitive hemopoietic progenitor cells.

High levels of the adhesion molecule CD44 on leukemic cells generate acute myeloid leukemia relapse after withdrawal of the initial transforming event

Multiple genetic hits are detected in patients with acute myeloid leukemia (AML). To investigate this further, we developed a tetracycline-inducible mouse model of AML, in which the initial transforming event, overexpression of HOXA10, can be eliminated. Continuous overexpression of HOXA10 is required to generate AML in primary recipient mice, but is not essential for maintenance of the leukemia. Transplantation of AML to secondary recipients showed that in established leukemias, ∼80% of the leukemia-initiating cells (LICs) in bone marrow stopped proliferating upon withdrawal of HOXA10 overexpression. However, the population of LICs in primary recipients is heterogeneous, as ∼20% of the LICs induce leukemia in secondary recipients despite elimination of HOXA10-induced overexpression. Intrinsic genetic activation of several proto-oncogenes was observed in leukemic cells resistant to inactivation of the initial transformation event. Interestingly, high levels of the adhesion molecule CD44 on leukemic cells are essential to generate leukemia after removal of the primary event. This suggests that extrinsic niche-dependent factors are also involved in the host-dependent outgrowth of leukemias after withdrawal of HOXA10 overexpression event that initiates the leukemia.

HOXB4-induced self-renewal of hematopoietic stem cells is significantly enhanced by p21 deficiency

Enforced expression of the HOXB4 transcription factor and downregulation of p21Cip1/Waf (p21) can each independently increase proliferation of murine hematopoietic stem cells (HSCs). We asked whether the increase in HSC self‐renewal generated by overexpression of HOXB4 is enhanced in p21‐deficient HSCs. HOXB4 was overexpressed in hematopoietic cells from wild‐type (wt) and p21−/− mice. Bone marrow (BM) cells were transduced with a retroviral vector expressing HOXB4 together with GFP (MIGB4), or a control vector containing GFP alone (MIG) and maintained in liquid culture for up to 11 days. At day 11 of the expansion culture, the number of primary CFU‐GM (colony‐forming unit granulocyte‐macrophage) colonies and the repopulating ability were significantly increased in MIGB4 p21−/− BM (p21B4) cells compared with MIGB4‐transduced wt BM (wtB4) cells. To test proliferation of HSCs in vivo, we performed competitive repopulation experiments and obtained significantly higher long‐term engraftment of expanded p21B4 cells compared with wtB4 cells. The 5‐day expansion of p21B4 HSCs generated 100‐fold higher numbers of competitive repopulating units compared with wtMIG and threefold higher numbers compared with wtB4. The findings demonstrate that increased expression of HOXB4, in combination with suppression of p21 expression, could be a useful strategy for effective and robust expansion of HSCs.

RNAi screen identifies MAPK14 as a druggable suppressor of human hematopoietic stem cell expansion

We report on a forward RNAi screen in primary human hematopoietic stem and progenitor cells, using pooled lentiviral shRNA libraries deconvoluted by next generation sequencing. We identify MAPK14/p38α as a modulator of ex vivo stem cell proliferation and show that pharmacologic inhibition of p38 dramatically enhances the stem cell activity of cultured umbilical cord blood derived hematopoietic cells. p38 inhibitors should thus be considered in strategies aiming at expanding stem cells for clinical benefit.

Identification of the chemokine CCL28 as a growth and survival factor for human hematopoietic stem- and progenitor cells.

In an attempt to discover novel growth factors for hematopoietic stem and progenitor cells (HSPCs), we have assessed cytokine responses of cord blood (CB)-derived CD34(+) cells in a high-content growth factor screen. We identify the immunoregulatory chemokine (C-C motif) ligand 28 (CCL28) as a novel growth factor that directly stimulates proliferation of primitive hematopoietic cells from different ontogenetic origins. CCL28 enhances the functional progenitor cell content of cultured cells by stimulating cell cycling and induces gene expression changes associated with survival. Importantly, addition of CCL28 to cultures of purified putative hematopoietic stem cells (HSCs) significantly increases the ability of the cells to long-term repopulate immunodeficient mice compared with equivalent input numbers of fresh cells. Together, our findings identify CCL28 as a potent growth-promoting factor with the ability to support the in vitro and in vivo functional properties of cultured human hematopoietic cells.

Robotic implementation of a microchip-based protein clean-up and enrichment system for MALDI-TOF MS readout

A capillary filling microfluidic proteomic sample processing system has been realized in an automated set-up. Chip-integrated solid-phase extraction is performed in a 96-array format followed by sequential capillary action elution into a piezoelectric microdispenser and subsequent transfer to MALDI targets. Samples are eluted and deposited in volumes of 200–300 nl. The robotic system offers calibration to user-defined microextraction arrays and MALDI-target formats at micrometre resolution. Built-in force feedback control ensures a precise and robust microchip docking/handling in three dimensions. An efficient automated washing protocol eliminates analyte carry-over. System throughput ranges typically from 50–100 samples h-1.

Implementation of a protein profiling platform developed as an academic-pharmaceutical industry collaborative effort

As much attention has devoted to the proteome research during the last few years, biomarker discovery has become an increasingly hot area, potentially enabling the development of new assays for diagnosis and prognosis of severe diseases. This is the field of research interest where efforts originating from both academic and industrial groups should jointly work on solutions. In this paper, we would like to demonstrate the fruitful combination of both research domains where the scientific crossroads sprout fresh ideas from the basic research domain and how these are refined and tethered to industrial standards. We will present an approach that is based on novel microfluidic devices, utilizing their benefits in processing small‐volume samples. Our biomarker discovery strategy, built around this platform, involves optimized samples processing (based on SPE and sample enrichment) and fast MALDI‐MS readout. The identification of novel biomarkers at low‐abundance level has been achieved by the utilization of a miniaturized sample handling platform, which offers clean‐up and enrichment of proteins in one step. Complete automation has been realized in the form of a unique robotic instrumentation that is able to extract and transfer 96 samples onto standard MALDI target plates with high throughput. The developed platform was operated with a 60 sample turnaround per hour allowing sensitivities in femtomol regions of medium‐ and low‐abundant target proteins from clinical studies on samples of multiple sclerosis and gastroesophageal reflux disease. Several proteins have been identified as new biomarkers from cerebrospinal fluid and esophagus epithelial cells.