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Stem cell mobilization in sickle cell patients
The patients were carefully monitored in the Intensive Care Unit and a few hours before the procedure started, they were given prophylaxis for vaso-occlusive crisis [hyperhydration 60 mL/kg/day of a 0.9% saline solution and oxygen therapy (2 L/min)], as recommended by French guidelines.21 These guidelines recommend oxygen therapy to prevent sickling phenomena in particularly stressful conditions. As mobilization can be consid- ered stressful, we decided to treat all patients with oxygen thera- py. Baseline O2 saturation levels were normal and remained stable during the procedure. The patients received a subcutaneous injec- tion of 0.24 mg/kg of Plerixafor. We then monitored whole blood HbS level, plasma bilirubin, conjugated bilirubin and lactate dehy- drogenase levels, and white blood cell, neutrophil and monocyte counts for 30 days after the Plerixafor injection. Plerixafor-mobi- lized HSPC in peripheral blood were collected by using a COBE® Spectra Optia apheresis system (Terumo BCT, Lakewood, CO, USA) with modifications (Online Supplementary Material and Methods). The apheresis lasted from 4 to 6 h. Product volumes and total blood volumes are indicated in Table 2. Egress of the CD34+ cells was monitored hourly for at least 24 h after the Plerixafor injection: total CD34+/kg collected and CD34+ collection efficiency (CE1) [total CD34+ collected/[L processed x (CD34+ pre + CD34+ post)/2] were evaluated following apheresis (Table 2).
Flow cytometry analysis
Cells were stained with specific antibodies (Online Supplementary Table S2) and analyzed on a BD FACSCantoTM II sys- tem, with gating on viable, 7AAD-negative cells. The data were processed using FlowJo software (version 10.2, Treestar, Ashland, OR, USA).
RNA-Seq extraction
Total RNA was extracted from 0.1-1x106 CD34+ cells using an RNeasy Micro kit (QIAGEN). RNA-Seq libraries were prepared from ~10 ng of total RNA, using the Ovation Human FFPE RNA-Seq Multiplex System kit (Nugen) after DNase treatment (ArcticZymes) and >40 million paired-end reads/sample. Samples were generated on a HiSeq 2500 instrument (Illumina). RNA-Seq analysis was per- formed as described in the Online Supplementary Methods.
Transplantation into non-obese diabetic severe combined immunodeficiency gamma mice
The non-obese diabetic severe combined immunodeficiency gamma (NSG) mice (NOD.CgPrkdcscid Il2rgtm1Wj/SzJ, Charles
Table 2. Characteristics of apheresis and CD34+ immunoselection.
River Laboratories) were housed in a pathogen-free facility. All experiments were performed in compliance with the French Ministry of Agriculture’s regulations on animal experiments, and were approved by the regional Animal Care and Use Committee (APAFIS#2101-2015090411495178 v4). Six- to 8-week-old mice were conditioned with intraperitoneally injected busulfan (Sigma, 25 mg/kg body weight/day) 72 h, 48 h and 24 h before transplan- tation. CD34+ cells (300,000 cells/mouse) from Filgrastim-mobi- lized healthy donors or Plerixafor-mobilized cells from the three SCD patients were transplanted into the NSG mice via retro- orbital sinus injection. Neomycin was added to the animals’ acid- ified drinking water. Engraftment was analyzed as described in the Online Supplementary Methods.
Statistical analysis
Statistical tests are indicated in the Figure legends. The thresh- old for statistical significance was set at P<0.05.
Results
Patients’ characteristics
Four SCD homozygous patients followed at Necker- Enfants Malades hospital and meeting our inclusion crite- ria were enrolled between May 2015 and January 2017. The first one was excluded from the study during the enrollment stage because of elevated granulocyte counts exceeding the limits established in the protocol (<10x109 granulocytes/L). The other three patients (P1 - P3) had been monitored in a reference center after the diagnosis of SCD within the first 4 years of life. All suffered from severe SCD, with a history of acute chest syndrome and more than two vaso-occlusive crises per year requiring hospitalization. P1 had undergone cholecystectomy and tonsillectomy. P2 had papillary necrosis and osteonecrosis of both femoral heads. P3 had undergone cholecystecto- my, and had chronic asthma and a history of osteomyelitis events. P1 and P2 were transfused monthly because years of hydroxyurea treatment had proven to be ineffective. Hydroxyurea treatment was stopped in P3 3 months before mobilization. P3 was then transfused monthly until mobilization. In view of iron overload caused by the transfusions (Table 1), treatment with deferasirox (P1) and deferiprone (P2) was ongoing.
Apheresis
Total blood volumes in liters (liters processed by the apheresis device)
Product volumes (mL)
Hematocrit value (%)*
CE1**
Total CD34+ cells collected by apheresis x106 Total CD34+ cells collected by apheresis x106/kg of body weight
CD34+ cell product after immunoselection
Recovery
Purity post-selection
SCD Pler 1
3.27 (15.8)
278 4.8 0.24 354 4.6 (77)
82%
95%
SCD Pler 2
4 (21)
382 5.8 0.30 412 5.8 (71)
92%
SCD Pler 3
4 (17.9)
322 8.2 0.29 292 4.5 (65)
31%
94.7%
79.5%
*Normal value: 2-3%; **CE1 = total CD34+ collected/ [L processed X (CD34+pre+CD34+post)/2]
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