The human bone marrow harbors a CD45− CD11B+ cell progenitor permitting rapid microglia-like cell derivative approaches

STANDARDS, PROTOCOLS, POLICIES, AND REGULATIONS FOR CELL-BASED THERAPIES The human bone marrow harbors a CD45 − CD11B + cell progenitor permitting rapid microglia-like cell derivative approaches Andreas Bruzelius 1,2 | Isabel Hidalgo 1,3 | Antonio Boza-Serrano 4,5 | Anna-Giorgia Hjelmér 1 | Amelie Tison 1 | Tomas Deierborg 5 | Johan Bengzon 1 | Tania Ramos-Moreno 1,5 1 Division of Neurosurgery, Department of Clinical Sciences Lund, Skåne University Hospital, Lund Stem Cell Center, Lund, Sweden 2 Department of Experimental Medical Science and Lund Stem Cell Center BMC, Lund University, Lund, Sweden 3 Institution for Laboratory Medicine, Division of Molecular Hematology, Faculty of Medicine, Lund University, Lund, Sweden 4 Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia e Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocio/CSIC/ Universidad de Sevilla, Seville, Spain 5 Department of Experimental Medical Science, Experimental Neuroinflammation Laboratory, Bio Medical Center (BMC)., Lund University, Lund, Sweden Correspondence Tania Ramos-Moreno, PhD, Division of Neurosurgery, Department of Clinical Sciences, Lund University, Lund Stem Cell Center, BMC B10, SE-221 84 Lund, Sweden. Email: [email protected] Abstract Microglia, the immune sentinel of the central nervous system (CNS), are generated from yolk sac erythromyeloid progenitors that populate the developing CNS. Interestingly, a specific type of bone marrow-derived monocyte is able to express a yolk sac microglial signature and populate CNS in disease. Here we have examined human bone marrow (hBM) in an attempt to identify novel cell sources for generating microglia-like cells to use in cell-based therapies and in vitro modeling. We demonstrate that hBM stroma harbors a progenitor cell that we name stromal microglial progenitor (STR-MP). STR-MP single-cell gene analysis revealed the expression of the consensus genetic microglial signature and microglial-specific genes present in development and CNS pathologies. STR-MPs can be expanded and generate microglia-like cells in vitro, which we name stromal microglia (STR- M). STR-M cells show phagocytic ability, classically activate, and survive and phagocyte in human brain tissue. Thus, our results reveal that hBM harbors a source of microglia-like precursors that can be used in patient-centered fast derivative approaches. KEYWORDS bone marrow, common myeloid progenitor, human bone marrow, microglia, microglial precursor, microglia-like cell in vitro model, primitive myeloid progenitor, pluripotent stem cell 1|INTRODUCTION Microglia are generated from yolk sac erythromyeloid progenitors (EMPs) that populate the developing central nervous system (CNS) to become its resident macrophage. 1,2 Microglia play a key role in the brain development, ensure maintenance and function during adulthood, 3 and are involved in the onset and progression of CNS pathologies, including neurodegenerative and psychiatric disorders 4 as well as malignant brain tumors. 5 Microglia constitute a target for therapy. 6 Development of drugs tries to target microglia for Johan Bengzon and Tania Ramos-Moreno contributed equally as senior authors. Received: 21 March 2020 Accepted: 25 October 2020 DOI: 10.1002/sctm.20-0127 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press. 582 STEM CELLS Transl Med. 2021;10:582–597.wileyonlinelibrary.com/journal/sct3 controlling immunomodulation of the CNS environment 7 and also possesses a potential in cell therapy applications as microglia exhibit neuroprotection in ischemia 8 and are scavengers for amyloid β(Aβ)in Alzheimer's disease. 9 Presently, the potential therapeutic application of human microglia-like cells is limited by the lack of CNS microglial sources and fast and safe stem cell approaches for deriving microglia. Current methods of generating induced pluripotent stem cells (iPSCs) that subsequently can be differentiated to microglia-like cells are timeconsuming (≥4 months), have low efficiency, and are potentially associated with tumor risks when used for transplantation purposes. 10,11 The use of embryonic stem cells suffers also from ethical concerns and limited access, 12 whereas approaches using microglia-like cells derived from bone marrow monocytes bring functional differences that are associated to their unique roles in lesion formation in the curse of CNS degeneration. 13-15 Interestingly, a specific type of murine bone marrow-derived microglia-like cells has been reported to be able to populate CNS in disease and to express a yolk sac microglial signature. 16 In the present work, we have examined human bone marrow (hBM) in an attempt to identify the presence of a cell source for generating human microglia-like cells in fast and reliable derivative approaches. We demonstrate the presence of a nonhematopoietic cell population, herein named stromal microglial progenitors (STR-MPs), in the stromal compartment of the hBM. Single-cell gene analysis shows that this cell population shares a microglial genetic signature, can be cultureexpanded in mesenchymal stromal cell (MSC) cultures, and can generate microglia-like cells (stromal microglia [STR-M]) in vitro. The STR-M retain microglial markers in vitro, have phagocytic and immune polarization abilities and microglia-like cell morphology, and survive in an ex vivo human brain organotypic model. We thus provide an efficient, quick, and nondisruptive procedure to generate human functional STR-M, with the capacity to become polarized upon lipopolysaccharide (LPS) exposure and to phagocyte latex beads and Aβfibrils, for use in in vitro microglial modeling and/or cell-based therapies. 2|MATERIALS AND METHODS 2.1 |Procurement of hBM stromal cells Bone marrow was aspirated from the iliac crest of healthy donors (60 mL, n = 5, median age 24 years, range 21-35 years). All participants in the study gave a written informed consent. The procedure was approved by the Swedish local ethics committee in Lund (Regionala Etikprövningsnämden Lund [EPN], protocol Dnr2009/12), and all experimental protocols were performed according to EPN's guidelines. Bone marrow mononuclear cells were isolated by density gradient centrifugation (LSM 1077 Lymphocyte; PAA Laboratories, Thermo Fisher Scientific, Waltham, Massachusetts) similar to that previously described, 17,18 but no RosetteSep Human Mesenchymal Stem Cell Enrichment Cocktail was performed. PharmLyse was used to further eliminate red blood cells from the cell preparation. Stromal cells were allowed to attach to plastic and were regularly culture-expanded in MSC expansion medium (StemMACS MSC Expansion Media; Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with the broad-spectrum antibiotic Primocin (1:1000; InvivoGen, San Diego, California). 2.2 |Flow cytometry and cell sorting of STR-M- like cells Single-cell suspensions from the freshly isolated hBM of the five healthy donors (three female donors and two male donors aged between 21 and 35 years) were stained in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline [PBS; Gibco, Thermo Fisher Scientific] + 10% fetal bovine serum [Gibco] + 0.2 mM ethylenediamine tetraacetic acid [EDTA; Sigma-Aldrich, St. Louis, Missouri]) for 30 minutes at 4C with the following antibodies: Brilliant Violet 421-cluster of differentiation (CD) 73 (AD2) (1/200) and Alexa Fluor 700-CD90 (5E10) (1/200), both from Nordic BioSite, Täby, Sweden; phycoerythrin (PE)-CD105 (266) (1/200), PE-cyanine (Cy) 5- human leukocyte antigen-ABC isotype (HLA-ABC) (G46-2.6) (1/200), PE-Cy7-CD45 (H130) (1/200), Brilliant Violet 605-CD11b (1/200), fluorescein isothiocyanate (FITC)-CD14 (M5E2) (1/200), FITC-CD19 (HIB19) (1/200), FITC-CD34 (581) (1/200), and FITC-HLA-DR isotype (HLA-DR) (G46-6) (all from BD Biosciences, San Jose, California). Propidium iodide (P3566; Life Technologies, Thermo Fisher Scientific) at a concentration of 1/1000 in FACS buffer was used as a viability marker prior to acquisition. All cells were analyzed and sorted on a FACSAria IIu cell sorter (BD Biosciences). 2.3 |RNA sequencing extraction and single-cell gene analysis Single HLA-DR − CD14 − CD19 − CD34 − CD45 − CD11b + cells were sorted into 4 μL lysis buffer. 19 Preamplification was performed using TaqMan primers and Taq/SSIII reaction mix (Invitrogen, Thermo Fisher Scientific). Linearity control and negative controls were included in each plate. Preamplification was performed according to a Significance statement The authors believe that the discovery of an intermediate specified myeloid microglia progenitor will open the way for developing novel patient-centered approaches and in vitro modeling. The use of stromal microglial progenitor as the starting point in derivative microglia-like cell approaches, instead of a somatic cell to first generate human pluripotent stem cells, reduces the risk of teratoma formation and the overall waiting time of current microglia. Thus, these results will be of broad general interest to a number of high-profile areas of research, including stem cells and in vitro modeling, neurodegeneration, neuropsychiatry, brain tumors, bone marrow stromal cells, hematopoietic system, and human ontogeny. STROMAL MICROGLIAL PROGENITOR IN HUMAN BONE MARROW 583 published polymerase chain reaction (PCR) protocol 19 with an extended 50C cycle. Complementary DNA was added to 48.48 Dynamic Array Chips (Fluidigm, South San Francisco, California) with individual TaqMan assays and quantitative PCR, analyzed on BioMarkHD (Fluidigm). 2.4 |Data analysis Quantitative PCR data were analyzed in the BiomarkHD analysis software (quality threshold 0.60, automatic global cycle threshold, and linear derivative baseline correction). After excluding controls, data were preprocessed in SCExV 20 where cycle threshold values were inverted, normalized to median expression of each cell, and z-score normalized. Unsupervised clustering analysis was performed using random forest clustering 21 and principal component analysis, and correlations were measured using the Spearman rank method. 2.5 |Cell culture of hBM MSCs and nonhematopoietic CD11b + cells Procured hBM MSCs were expanded with StemMACS MSC Expansion Media (MiltenyBiotec) supplemented with Primocin (1:1000, InvivoGen), which we refer to as expansion or basal medium (BM) interchangeably, in T75 plastic flasks (Thermo Fisher Scientific). When they reached confluence, cells were trypsinized using trypsin- (0.25%), phenol red (Thermo Fisher Scientific). 2.6 |hBM MSCs and nonhematopoietic CD11b + cell expansion in serum-containing and serum-free conditions For serum-containing experiments: When they reached 90% confluence in T75 plastic flasks (Thermo Fisher Scientific), cells were kept in their StemMACS MSC Expansion Media (MiltenyBiotec) supplemented with Primocin (1:1000, InvivoGen) for 6 days, mimicking the serum-free medium experiments (described in the paragraph below). Next, preconditioned cells were seeded in 24 multiwells onto laminin (Sigma-Aldrich) and polyornithine (Advanced BioMatrix, Carlsbad, California) coated coverslips at a density of 80 000 to 100 000 cells per well. Next day after the seeding, cells were exposed to (a) only this expansion medium, BM; (b) BM supplemented with brainderived neurotrophic factor (BDNF) and human neutrophin 3 (NT3) (both from PeproTech, Rocky Hill, NJ; final concentration of 10 ng/mL) added fresh every second day for as long as required by the time points (2 or 4 weeks; Figure S1B); or (c) BM supplemented with macrophage colony-stimulating factor (M-CSF; 25 ng/mL; ThermoFisher Scientific), interleukin (IL)-34 (100 ng/mL; PeproTech), and transforming growth factor (TGF) β-1 (50 ng/mL; Militenyi) added fresh every second day for as long as required by the time points (2 or 4 weeks; Figure S1B). For serum-free medium experiments:Experimentalprotocolstarted when cells reached 90% confluence in the T75 flasks; cells were then exposed to base medium consisting of Dulbecco's modified Eagle's medium/F12 (Gibco), 1×N2 (Gibco), 1×Non-essential amino acids (NEAA;Gibco), and 1×B27 (Gibco), and supplemented with Primocin (1:1000, InvivoGen) for 6 days. Next, these preconditioned cells were seeded in 24 multiwells with coated coverslips at a density of 80 000 to 100 000 cells per well and treated as before, using the serum-free medium, which we refer to as neuronal medium (NM), supplemented with growth factors or cytokines (CKs) for 2 or 4 weeks (Figure S1B). 2.7 |Immunocytochemistry After fixing with an ice-cold paraformaldehyde 4% solution diluted in a 0.1 M potassium phosphate-buffered solution (4% paraformaldehyde [PFA]) for 30 minutes at room temperature, cells were permeabilized with 0.25% Triton X-100 for 15 minutes at room temperature. The cells were then blocked using donkey serum (1:20) for 30 minutes at room temperature before incubating with primary antibody for 2 hours and then with secondary donkey antibody for 1 hour coupled to either Cy3, Alexa Fluor 488, or Alexa Fluor 647 for anti-chicken, goat, mouse, or rabbit IgG antibodies (all from Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania; 1:200). Between each step the cells were washed with PBS three times for 5 minutes. Primary antibodies used were against the human CD11b (Abcam, Cambridge, Massachusetts; 1:200; rabbit), human ionized calcium-binding adapter molecule 1 (Iba1; Abcam; 1:200; goat), human transmembrane protein 119 (TMEM119; Abcam; 1:200; rabbit), fractalkine or chemokine ligand 1 (CX3CR1; Abcam; 1:200; mouse), human CD33 (Abcam; 1:200; mouse), major histocompatibility class II HLA-DR (Abcam; 1:200; mouse), and green fluorescent protein (GFP; Abcam; 1:200; chicken). As negative control the primary antibody was omitted. All antibodies used in the present work are listed in Table S6A. 2.8 |Counting Quantification analysis was done in an automated image analysis using the software Thermo Scientific HCS Studio: Cellomics Scan, version 6.6. A manual counting was added to exclude possible artifacts. The threshold levels were set to an internal control that contained only secondary antibodies. The high intensity levels were calculated per well as equal to or higher than the mean for the intensities plus twice the SD. Quantification of cells derived from hBM MSCs was done manually after taking images at ×20 magnification using the filter for 40,6-diamidino-2-phenylindole U-MNUA2 (excitation filter 365/5), tetramethylrhodamine/Cy3 49 005 (excitation filter band pass 454/ 15), and Cy5 49 009 (excitation filter band pass 640/15) under the epifluorescence microscope (Olympus, Tokyo, Japan) and using the software CellSens. A total of 10 visual fields per coverslip was used for cell counting, and a total of 50 to 100 cells per coverslip were counted for every condition using the ImageJ software. The high 584 BRUZELIUS ET AL. intensity levels were calculated by manually comparing the intensities between cells per field. 2.9 |Microglial phagocytosis assay Fluorescence latex beads of 1 μm diameter (Sigma-Aldrich) were first preopsinized for 1 hour at 37C at a ratio of 1:5 with fetal bovine serum (Gibco). Preopsinized beads were subsequently diluted in the different serum-containing and serum-free culture conditions for a final concentration of latex beads of 0.01% (Figure S2A). Cells were in contact with medium and preopsonized latex beads for 1 hour at 37Candsubsequently fixed with cold 4% PFA solution for a posterior immunocytochemistry study for CD11b and Iba1 expression. 2.10 |Aβoligomer preparation and treatment Aβ(M1-42) fluorescent peptides (wavelength 555) were prepared by diluting Aβdimethylsulfoxide stock at a final 186 μM in PBS by incubating it at 37C for 24 hours. This stock was brought to a final concentration of 0.5 μM in NM supplemented with CKs (NM + CK) or NM supplemented with neutrophins (NM + NT) and left over night (O/N) at 4C to allow the formation of fibrils and oligomers before completely changing the medium of the cells. Presorted nonhematopoietic CD11b + cells were seeded in a 96 multiwell, at a density of 50 000 cells per well, and, after being exposed to either NM + CK or NM + NT for 6 days, media containing the Aβ42 oligomers and cells were incubated for 1 hour at 37C, respectively, and fixed in cold 4% PFA for posterior analysis. 2.11 |LPS induced activation Presorted nonhematopoietic CD11b + cells were seeded at a density of 80 to 100 ×10 3 cells per well in 24 multiwell plates and treated as depicted in the scheme (Figure S1B). Cell cultures were incubated for 24 hours with 100 ng/mL of LPS from Escherichia coli O111:B4 (Sigma- Aldrich) added to the different serum-free culture conditions (Figure S2B). After 24-hour incubation, supernatants were collected, frozenindryice,andstoredat−80C for a subsequent Meso Scale analysis. 2.12 |Meso Scale The release of proinflammatory CKs in culture media upon LPS activation was measured using Meso Scale (Meso Scale Diagnostics, Rockville, Maryland) plates with the proinflammatory panels for interferon-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor-α. The plates were developed using the 4×reading buffer diluted to a factor of 1×with distilled water, and the plates were read using the QuickPlex Q120 reader from Meso Scale. The detection ranges of the different CKs measured were as follows: IL-1β (1670-0.408 pg/mL), IL-4 (1660-0.405 pg/mL), IL-12 (32200-7.86 pg/ mL), IL-10 (3410-0.833 pg/mL), interferon-γ(938-0.229 pg/mL), IL-2 (2630-0.642 pg/mL), IL-5 (967-0.236 pg/mL), IL-6 (5720-1.40 pg/mL), KC/GRO (1980-0.483 pg/mL), and tumor necrosis factor-α (627-0.153 pg/mL). 2.13 |Lentiviral transduction and antibiotic selection of human stromal cells hBM MSCs along with the nonhematopoietic CD11b + cells were transduced at passages 4 and 5 in T75 flasks at 80% confluence. Medium was completely changed with 10 mL of the StemMACS MSC Expansion Media (Milteny Biotec) containing 10 μL lentiviral construct (1.7 ×10 8 ) carrying a noninducible construct for GFP with puromycin resistance (73 582; Addgene, Watertown, Massachusetts). Preparations of lentiviral particles were produced according to protocol from 22 in a biosafety level 2 environment by the Vector Core Facility at the Lund Stem Cell Center. Next day after lentiviral transduction, medium was changed back to StemMACS MSC Expansion Media (Milteny Biotec) supplemented with Primocin (1:1000, InvivoGen). Forty-eight hours after infection, cultures were treated with puromycin at a final concentration of 10 μg/mL for 5 days. 2.14 |Coculture of human nonhematopoietic CD11b + cells, stromal cells, and adult human cortex organotypic slice cultures Adult human cortical tissue from three patients with glioma was obtained by informed consent from the patients according to guidelines approved by the Lund-Malmö Ethical Committee. All three patients were male and aged 52 to 66 years, and tumors were located in the frontotemporal region in these cases. Tumor pathology varied: isocitrate dehydrogenase (IDH) wild-type primary glioblastoma, IDH wild-type secondary glioblastoma, and IDH-mutated diffuse astrocytoma. In all cases, tissue for culturing was collected from magnetic resonance imaging (MRI)-positive, macroscopically pathological parts of the tumor front. The surgically resected tissue was placed in ice-cold solution of artificial cerebrospinal fluid solution (119 mM NaCl, 26 mM NaHCO 3 , 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 1.3 mM MgSO 4 , and 11 mM glucose dissolved in Milli-Q and bubbled with carbogen for 15 minutes before adding 2.5 mM CaCl 2 ) and transferred to the slicing chamber of a vibratome (VT1200S; Leica, Wetzlar, Germany) where 250-μm thick tissue slices were cut. The sliced tissue was transferred to prepared six-well plates with equilibrated culture medium and cultured at 37C in a humidified atmosphere of 5% CO 2 . Organotypic tissue slices were cultured on Alvetex Strata six-well membrane inserts (AMS Biotechnology, Milton, UK) that had been presoaked in organotypic culture medium (neurobasal, 2% B27 without vitamin A, 0.5% Glutamax, and 10 μg/mL gentamicin; all from Thermo Fisher Scientific) in six-well plates, for a total of 3 weeks, and every second day the inserts were transferred to a new plate with 6 mL fresh and equilibrated medium in similar fashion as previously reported by Miskinyte et al. 23 STROMAL MICROGLIAL PROGENITOR IN HUMAN BONE MARROW 585 At day 7, one well per medium was selected and grafted with GFP + human predifferentiated nonhematopoietic CD11b + as part of the hBM MSC cultures. Briefly, 10 μL of a suspension mix (1 000 000 GFP + hBM MSCs) was collected into a cold glass capillary and injected as small drops stabbing the semidry slices at various sites in a randomized fashion trying to cover most of the surface. Slices were transplanted with 10 μL of suspension leading to approximately 300 000 cells per slice. Additional medium was added 30 minutes later to fully immerse the organotypic culture. The medium was changed every other day during the week, and coculture was maintained for 3 weeks before fixation. Brain tissue was fixed in 4% PFA solution, washed three times with PBS, and submerged in sucrose at an increase of 10% each day for 3 days, with the final day being 30%, and subsequently stored in antifreeze solution (30% ethylene glycol and 30% glycerol, both from VWR International, West Chester, Pennsylvania) in 0.012 M NaH 2 PO 4 H 2 O and 0.031 M Na 2 HPO 4 2H 2 O (both from Sigma-Aldrich) at −20C (Figure S2C). 2.15 |iDISCO Immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO) was performed using the readily available protocol at https://idisco.info/idisco-protocol/ with slight modifications. In brief, samples were fixed with 4% PFA and prepared using the methanol pretreatment; samples were then incubated in permeabilization solution for 1 day followed by blocking solution for 1 day. Samples were incubated with the primary antibodies anti GFP (Abcam; 1:200; chicken), human CD11b (Abcam; 1:200; mouse), and TMEM119 (Abcam; 1:200; rabbit) and with secondary antibodies Cy3, Alexa Fluor 488, or Alexa Fluor 647 for anti-chicken, goat, mouse, or rabbit IgG antibodies (all from Jackson ImmunoResearch Laboratories; 1:200); incubation was set to 2 days with washing steps between for 1 day. Succeeding clearing steps were done following the recommended times in the protocol. Samples where then mounted with PO-PRO-1 Iodide (Invitrogen) mixed with DABCO and imaged using a Zeiss LSM 780 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). 2.16 |Functional assays in the cocultures and histology For the functional assays in the cocultures, adult human cortical tissue from one patient with glioma was obtained, sliced, and brought on the inserts as described in the previous section. Three to 5 days after slicing, a total of 30 000 STR-MP cells per slice previously sorted were labeled with the fluorescent vital dye CellTracker Red CMTPX (Thermo Fisher Scientific) according to the manufacturer's instructions and were grafted the same day in similar fashion as described above. Cocultures were kept for 6 days in NM + CK (prepared as described above), and medium was changed every 3 days to a previously equilibrated NM + CK. At this time point, latex beads were added into NM + CK with LPS from E. coli O111:B4 (Sigma-Aldrich) at a 100 ng/mL concentration, and the media of the cocultures were changed to this one. After 24 hours, cocultures were fixed in cold 4% PFA for 20 minutes and left in PBS for further histological analysis. Previous to image acquisition, cocultures were treated O/N at 4C with a 1% Triton X-100 in PBS permeabilizing solution containing PO-PRO-1 Iodide (1:200, Invitrogen). 2.17 |Statistical analysis All statistical analysis was performed using Graph Pad Prism version 7.0c. Data were assumed to fit a normal distribution, and statistical comparisons were made using unpaired, two-tailed ttests for comparisons of two means between untreated groups and/or for comparisons between different time points within a condition; onetailed unpaired ttest when comparing two groups exposed to a treatment, that is, LPS 24 ; and, depending on the number of variables, one- or two-way analysis of variance (ANOVA) followed by Tukey's test when comparing multiple groups. A value of P<.05 was considered statistically significant. Data are presented as means ± SEM. 3|RESULTS 3.1 |Identifying STR-MP cells We hypothesized that, because the adult hematopoietic system derives from the hematopoietic stem cells originated during the definitive hematopoiesis, 25,26 if EMPs or EMP-like cells are present in the adult hBM, they are likely to be part of the stromal fraction. In order to target the stromal fraction and avoid genetic drift, selection bias, and enrichment of specific populations in the samples, fresh isolated hBM was analyzed using flow cytometry (Figure 1A), initially to negatively select monocyte/macrophages. Within the remaining fraction, CD14, CD19, CD34, and HLA-DR cell surface markers were used to remove hematopoietic and endothelial cells in the bone marrow extractions (Figure 1B). The absence of the above-mentioned markers together with CD45 was used to help to identify the hBM stromal compartment 27 from which CD11b + cells were selected, with HLADR − CD14 − CD19 − CD34 − CD45 − CD11b + cells constituting 0.04% ± 0.01% of hBM cells (n = 5; Figure 1B). Of the STR-MP cells, 97.9% ± 1.1% were negative for CD73 (Figure 1C), 99.8% ± 0.1% were negative for CD90 (Figure 1D), 97.7% ± 1.1% were negative for CD105 (Figure 1E), and 82.7% ± 9.2% were negative for HLA-ABC (Figure 1F), indicating the presence of an expected CD45 − CD11b + human microglia-like/myeloid progenitor profile 28,29 in the stromal fraction that does not share the mesenchymal molecular signature. 27 Sorted CD45 − CD11b + stromal cells were subjected to single-cell analysis for essential, disease-involved, and developmental microglialspecific genes, as well as for the consensus microglial signature. The sorted cells expressed the typical microglial progenitor gene expression profile (Figure 1G; Table S1), including the myeloid-associated transcription factors Runt related transcription factor 1 (RUNX1), Spi- 1 proto-oncogene (PU.1) and Colony stimulation factor 1 receptor 586 BRUZELIUS ET AL. FIGURE 1 Legend on next page. STROMAL MICROGLIAL PROGENITOR IN HUMAN BONE MARROW 587 (CSF1R), which are essential for regulation of microglial cell development, 1,30 and the microglia-enriched protein TMEM119 and beta-hexosaminidase subunit beta (HEXB), indicating an early commitment toward microglial fate. 31,32 The STR-MP single-cell gene profile showed a canonical microglial gene expression pattern, including TREM2,P2RY12,CD33,GPR34,GPR56,C1Q,CABLES1,BHLHE41, TMEM119,TGFBR1,ENTPD1,ITGB2,ITGAM,AIF,IRF8,ADORA3, and PPARD. 30,31,33–35 Because the vast majority of the microglial core gene signature is shared between immature progenitors and more mature microglial cells, 36 our data suggest that a myeloid/microglialike progenitor cell compartment is present in the hBM stromal compartment, which we refer to as the STR-MP compartment. 3.2 |Culturing STR-MP cells In order to study if STR-MP cells could derive microglia-like cells in vitro, we first tried to find the optimal culturing conditions for STRMP. Eight attempts using five different donors for isolating STR-MP from hBM samples showed that, unless reaching a minimum density of 30 000 STR-MP cells per well in a 96 multiwell-plate (76.93 mm 2 ), STR-MPs soon become apoptotic or quiescent when in contact with plastic (data not shown), suggesting that STR-MP cells could benefit from a supportive cell layer for survival, as STR-MP cells' profile was detected upon passages in the stromal cultures (Figure S1A). In a first attempt to follow and distinguish STR-MP cells within the hBM stromal cultures, fresh hBM stromal samples were divided in two. Half of the samples were frozen, and the other half was expanded on plastic to be infected with a lentiviral vector coding for GFP followed by puromycin resistance on passage 4. One day after infection, cells were subjected to a subsequent 5-day puromycin selection. Next, the frozen fresh bone marrow stromal cells corresponding to the same hBM samples were used to sort STR-MP cells as described above, and the isolated STR-MP cells were seeded with the corresponding donor, now being GFP + and on passage 6 (Figures S1B and S2A). After 2 days, cells were transferred to serumfree medium to facilitate macrophage-like differentiation, 37 medium that was supplemented with neurotrophins (NTs) to promote hBM MSC survival and, putatively, the STR-M 38 (Figure S1B). Cells were fixed at day 9 for an immunocytochemical analysis. GFP − STR-MP CD11b + cells were detected both on plastic and in close contact with hBM stromal cells. Immunocharacterization of STR-MP CD11b + cells revealed high levels of Iba1 as well as TMEM119 expression (Figure 2B), indicating the maintenance of microglial marker previously detected on the single-cell gene analysis (Figure 1G). In order to find stimulatory conditions that can promote the presence of STR-MP cells in vitro, nontransduced bone marrow stromal cell cultures at low passages (less than passage 6; based on Yang et al 39 ) were subjected to different culturing conditions. The expansion medium containing serum was thus supplemented with either (a) NTs (human BDNF and NT3) or (b) CKs (IL-34, M-CSF, and TGF-β). The use of BDNF and NT3 (the NT condition) was expected to promote cell growth and survival of hBM MSCs and, putatively, the STR-M. 38,40 The use of IL-34, M-CSF, and TGF-β(the CK condition) was expected to facilitate culturing and differentiation into microglialike cells, as described when using derivative approaches from human stem pluripotent sources. 41 Two time points of 3 and 5 weeks were included (Figure S1C) when cells were fixed for morphological and immunocytochemical studies of a combination of microglial markers as follows: Iba1 and CD11b, Iba1 and TMEM119, TMEM119 and HLA-DR, and Iba1 and CX3CR1. Images were analyzed using the software ThermoScientific HCS Studio: Cellomics Scan, version 6.6 (see Section 2). Two distinct populations defined by their Iba1 immunoreactivity and nuclear area size were observed under the different serum-con- taining conditions (Figure S1D and Table S1A). Iba1 + CD11b + cell nuclei area was restricted to 100 to 400 squared pixels (40-156 μm 2 ); thus, in subsequent analyses, we restricted counting to immunoreactive CD11b + cellswithanucleussizeequaltoorless than 400 squared pixels (156 μm 2 ). Within this group of small cells, CK conditions show an increase in the proportion of Iba1 + CD11b + cells below 100 squared pixels in the stromal cell cultures with time (Figure 2C; 0.26% ± 0.12% at the 3-week time point vs 0.81% ± 0.09% Iba1 + CD11b + cells at the 5-week time point in BM supplemented with CKs [BM + CK]; one-tailed unpaired ttest, P≤.05; n = 2 donors). Expression of microglial markers revealed that, on average and at 5 weeks under the same BM + CK condition, 59.16% ± 34.16% of the small cells are positive for TMEM119, 98.91% ± 1.08% are CX3CR1 + , and 87.5% ± 12.5% do not express HLA-DR. This pattern repeats across the different serum conditions, and the proportions do not vary between them, suggesting that this cell type and its expression pattern is maintained under all conditions (Figure 2D and Table S1B-E). A serum-free medium condition was also included to closely mimic the CNS environment, 42 facilitate macrophage-like differentiation, 37 FIGURE 1 Human bone marrow stromal cells harbor nonhematopoietic CD11b + cells that express microglial-specific genes. A-F, Representative case showing the gating strategy for detecting nonhematopoietic CD11b + cells. Cells negative for markers HLA-DR, CD14, CD19, and CD34 that label endothelial and hematopoietic lineages and hematopoietic stem cells were selected within the compartment of living cells. Selected cells (HLA-DR − CD14 − CD19 − CD34 − /CD45 − CD11b + ) were analyzed for CD90, CD73, CD105, and HLA-ABC mesenchymal markers (n = 5 donors). G, Single-cell gene analysis in the CD45 − CD11b + nonhematopoietic nonendothelial compartment reveals a microglial expression pattern. Analyzed genes include the microglial consensus signature, genes involved in microglial development and maturation, and genes reported to be misfunctional in neurodegenerative and psychiatric disorders (n = 4 donors). Each column represents the gene analysis for a sorted cell, whereas the y-axis provides a list of the genes. CD, cluster of differentiation; FSC-A, forward scatter-area; FSC-H, forward scatter-height; HLAABC, human leukocyte antigen-ABC isotype; PI, propidium iodide; SSC-A, side scatter-area 588 BRUZELIUS ET AL. and study a possible enhancement effect when supplementing with NTs or CKs. This study could not be completed as cells were not viable without serum. Hence, the use of serum and a feeder cell layer such as the hBM stromal cells is highly recommended for an optimal expansion of the STR-MP cells. The use of CK can promote the STR-MP compartment by increasing their proportion in the stromal cultures upon time of exposure, and STR-MP cultured cells retain the expression of microglial markers, that is, CD11b, Iba1, TMEM119, and CX3XR1. FIGURE 2 Legend on next page. STROMAL MICROGLIAL PROGENITOR IN HUMAN BONE MARROW 589 3.3 |Long-term culturing of STR-MP cells The clonal complexity of MSCs vary upon cell expansion and is believed to depend on the selection of dominant MSC clones. 43 In order to address if cell expansion affects the fate of STR-MP so that the STR-MP cell clone becomes dominant and its abundance in vitro is promoted upon passages, hBM stromal cultures were first expanded until apparent morphological changes due to passaging were observed (after passage 6, Figure S1E,F, based on Yang et al 39 ). Cells were collected and stained for the cocktail of markers previously used for FACS and analysis in fresh bone marrow samples. The proportion of STR-MP at high passages compared with the proportion of STR-MP from fresh samples showed that in vitro cell expansion promotes the presence of STR-MP cells in culture (0.04% ± 0.01% of STR-MP in fresh sample vs 5.74% ± 3.05% of STR-MP in cell cultures after passage 6, unpaired two-tailed Student's ttest, n = 5, P< .05; Figure 2E). We next addressed the influence of in vitro long-term culturing on the STR-MP cellular characteristics, as functionality and differentiation of cultured cells can be affected and are important to consider for stem cell-based therapies. 39 Our results show that the use of NT in serum-containing medium significantly increased Iba1 + CD11b + cell numbers when compared with the CK-supplemented medium at 3 weeks (Figure 2F and Table S2; 46.96 ± 11.15 BM supplemented with neutrophins vs 8.42 ± 6.63 BM + CK; unpaired, one-tailed Student's ttest, P≤.05; n = 2 donors; passage numbers 7-8) but were significantly reduced to zero at 5 weeks in all conditions (Figure 2G and Table S2A-D), and, similar to the low passaging cell cultures, STR-MP high passaging cells defined by their expression on Iba1 and CD11b retain the expression of microglial markers (Figure 2G,H). Long-term cultured Iba1 + CD11b-high cells were also able to survive under serum-free condition (Figure 2H-K and Table S2E-H). In serum-containing medium, the expression of TMEM119 can vary between 34.06% ± 24.31% and 74.15% ± 4.61% of Iba1 + cells, whereas in serum-free medium the percentage of Iba1 + cells expressing TMEM119 varies between 51.52% ± 22.94% and 86.67% ± 2.21%. In serum-free and serum-containing conditions, all Iba1 + cells express CX3CR1 (Table S2G). Regarding the absence of HLA-DR, between 80.85% ± 19.14% and 94.35% ± 5.6% of TMEM119 + cells do not express HLA-DR in serum-supplemented medium, and over 99.04% ± 0.96% lack HLA-DR expression in serum-free medium (Figure 2H and Table S2H). Thus, STR-M defined as Iba1 + CD11b-high cells in stromal cell cultures can survive in both serum-containing and serum-free conditions for up to 5 weeks. Moreover, STR-MP cells significantly increase their numbers upon exposure of NT in a serum-containing medium for 2 weeks and retain the expression of microglial markers but are not detected after this time. FIGURE 2 STR-MP CD11b + cells grow as part of mesenchymal stromal cultures, which act as a feeder cell layer. A, Twelve hours after sorting, sorted cells were seen attaching to plastic (a) showing a cubic cellular shape (denoted by an asterisk; scale bar, 20 μm; magnification in the upper frame to the right (a0); scale bar, 10 μm). In order to force a morphological change, cells were exposed to a serum-free medium supplemented with neurotrophins to promote survival of human bone marrow-derived mesenchymal stromal cells. After 2 days of serum-free conditions, different ramified morphologies could be observed (b) (asterisk; scale bars, 20 μm; n = 2 donors, two repetitions). B, Cells were able to survive for 5 days on plastic and in contact with stromal cells. After 1 week of serum-free condition exposure, cells were fixed in 4% and analyzed for Iba1 and TMEM119 expression. The arrow denotes a small GFP − CD11b + cell denoted by Hoechst and immunoreactive toward Iba1 and TMEM119. The asterisk shows a small GFP − CD11b + cell immunoreactive to Iba1 and negative to TMEM119 expression (scale bar, 20 μm; n = 2 donors, two repetitions). C, Analysis of the small nuclei compartment. The condition BM + CK showed a significant increase on Iba1 and CD11b immunoreactive cells (n = 2 donors; two-way ANOVA; *P≤.05; **P≤.005; ***P≤.001). D, Expression of microglial markers Iba1, TMEM119, and CX3CR1 were included to determine if cells maintain their expression under the different culturing conditions. HLA-DR was included as a negative control (Table S1). E, Fluorescence-activated cell sorting analysis comparison of the STR-MP compartment between high passage number (after passage 6) and fresh bone marrow sample shows that the STR-MP compartment increases with passage number (0.04% ± 0.01% of STR-MP in fresh sample vs 5.74% ± 3.05% of STR-MP in cell cultures after passage 6, unpaired two-tailed Student's ttest, n = 5, P< .05). F, Stromal microglial CD11b + cells were detected based on CD11b and Iba1 coexpression. The graph shows the percentage of these cells compared across the different medium conditions and two time points, 3 and 5 weeks. A higher percentage of Iba1 CD11b immunoreactive cells was observed at 3 weeks under the serum medium supplemented with neurotrophins (n = 2 donors; one-way ANOVA; *P≤.05). G, The graph shows the percentage of cells in high passage cell cultures expressing the microglial markers Iba1, TMEM119, and CX3CR1 across culture serumcontaining conditions. HLA-DR was included as a negative control (Table S2). H, The graph shows the percentage of cells in high passage cell cultures under serum-free conditions for microglial markers Iba1, TMEM119, and CX3CR1. HLA-DR was included as a negative control (Table S2). Note that the legend depicting the different immunocyochemistries applies for D, G, and H. Also, all cells were CX3CR1 immunoreactive. I, Representative Iba1 and TMEM119 cell in high passage cultures in the serum-free supplemented with neurotrophins condition showing a microglial morphology at the 3-week time point. Scale bar, 20 μm. J, Representative Iba1 and CX3CR1 cells in high passage cultures in the serumfree supplemented with neurotrophins condition showing microglial morphologies at the 3-week time point. Scale bar, 20 μm. K, Representative TMEM119 and CD33 cell in high passage cultures in the serum-free supplemented with neurotrophins condition showing a microglial morphology at the 3-week time point (counting can be found in Table S2; n = 2 donors; one-way ANOVA; P> .05). Scale bar, 20 μm. All data presented as means ± SEM. BM, basal medium (expansion medium); BM + CK, basal medium supplemented with cytokines; BM + NT, basal medium supplemented with neutrophins; CD, cluster of differentiation; CX3CR1, fractalkine or chemokine ligand 1; DAPI, 40,6-diamidino-2- phenylindole; GFP, green fluorescent protein; HLA-DR, human leukocyte antigen-DR isotype; Iba1, ionized calcium-binding adapter molecule 1; NM, neuronal medium; NM + CK, neuronal medium supplemented with cytokines; NM + NT, neuronal medium supplemented with neutrophins; NT, neutrophin; STR-MP, stromal microglial progenitor; TMEM119 transmembrane protein 119 590 BRUZELIUS ET AL. 47. Wislet-Gendebien S, Laudet E, Neirinckx V, et al. Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences. Cell Mol Life Sci. 2012;69:2593-2608. 48. Treutlein B, Brownfield DG, Wu AR, et al. Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. 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