In Vitro Erythropoietic Response to Enzyme Replacement Therapy in Gaucher Type 1 Patients

Introduction. Gaucher disease type 1 (GD, OMIM #230800) is the most common inherited lysosomal storage disorder resulting from lysosomal glucocerebrosidase (GBA, acid-β-glucosidase) deficiency and subsequent accumulation of glucosylceramide in macrophages. This leads to anemia, thrombocytopenia and coagulopathy, visceral (hepatosplenomegaly, lungs) and skeletal manifestations (deformities, fractures, avascular osteonecrosis). Anemia is attributed to bone marrow infiltration by Gaucher cells, to inflammation and to the presence of splenomegaly; however the underlying pathophysiological mechanism hasn't been fully elucidated.

Aim. The aim of this study is to evaluate the in vitro erythroid status at baseline (T0) and the response to Enzyme Replacement Therapy (ERT) after 6 months (T1) and 1 year (T2). We evaluated the expansion and differentiation in erythroid cultures of CD34⁺ cells obtained from peripheral blood of GD type 1 patients at diagnosis before starting ERT (T0), at 6 months (T1) and at 1 year (T2) of ERT.

Methods. CD34⁺ cells were obtained by immunomagnetic separation from peripheral blood mononuclear cells of GD patients (n=3) and controls (n=5). Isolated CD34⁺ were evaluated by in vitro clonogenic capacity assays using the methylcellulose semisolid culture medium after 14-16 days of culture. Hematopoietic colonies (burst-forming unit-erythroid (BFU-E) and colony-forming unit-macrophage (CFU-M)) were scored with an inverted microscope. CD34⁺ cells were expanded in StemSpan medium, 1% StemSpan CC100 cytokine cocktail, 2 U/ml Erythropoietin (EPO) and 1 μM dexamethasone. Differentiation was induced in StemSpan medium with 10 ng/ml SCF and 10 U/ml EPO. Proliferation and surface marker expression (CD34, CD45, Glycophorine A, CD71) were examined by flow cytometry. Cell morphology was analyzed on cytocentrifuged smears stained with May-Grunwald-Giemsa.

Results. At T0 we observed a higher number of BFU-E in GD samples (126±23 BFU-E/10⁵ cells) compared to controls (49±12 BFU-E/10⁵ cells) (Tab.1). Similarly, higher macrophage CFU-M were observed in GD samples (29±12 CFU-M/10⁵ cells) compared to controls (10±1 CFU-M/10⁵ cells) (Tab.1). The BFU-E and CFU-M morphology showed no difference between GD patients and controls. The increased proliferation of erythroid precursors of GD patients was associated to impaired ability to differentiate in liquid cultures after 14 days (45,9% proerythroblasts and only 4% polichromatophilic erythroblasts) compared to controls (27% and 19%, respectively) (Tab.1). Flow cytometry confirmed an impaired differentiation of GD erythroblasts compared to controls at day 14 of the erythroid liquid cultures, as shown by high percentage of CD71^high/GlyA^low early erythroid precursor cells and low percentage of CD71^high/GlyA^high mature erythroblasts (GD: 73,9 and 4,9%, respectively; controls: 67,2 and 22.4%, respectively) (Fig.1). In samples from GD patients, after 1-year treatment with ERT (T2), we observed a reduction in the number of BFU-E compared to T0, reaching values similar to controls (GD T0: 126±23 BFU-E/10⁵ cells, T2: 41±11 BFU-E/10⁵ cells; controls: 49±12 BFU-E/10⁵ cells) and an increased number of mature erythroid cells generated in GD liquid cultures (Tab. 1). Also CFU-M were reduced at T2 compared to T0 (GD T0: 29±12 CFU-M/10⁵ cells, T2: 8±5 CFU-M/10⁵ cells; controls: 10±1 CFU-M/10⁵ cells) (Tab.1). An increased percentage of more differentiated erythroid cells at T2 compared to T0 was confirmed by flow cytometry in cultures from GD patients, as shown by low percentage of the CD71^high/GlyA^low early erythroid precursor cells and high percentage of CD71^high/GlyA^high mature erythroblasts on day 14 (GD: 44,7 and 45,6%, respectively; controls: 67,2 and 22.4%, respectively) (Fig.1).

Conclusions. These preliminary data suggest that anemia in GD might partly be related to a delay in erythroid differentiation. ERT treatment seems to correct the dyserythropoiesis by improving erythroid differentiation. Since glucosylceramide accumulates in macrophages, that are key elements in the erythroid niche, impaired erythroid differentiation might be related to the pathological micro-environment, rather than intrinsic hematopoietic stem cell defect. More extensive studies are needed to better understand the pathophysiology of anemia and pancytopenia of this rare disease.This study was partially supported by Sanofi Genzyme.

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