Retrieval of germinal zone neural stem cells from the cerebrospinal fluid of premature infants with intraventricular hemorrhage

Retrieval of germinal zone neural stem cells from the cerebrospinal fluid of premature infants with intraventricular hemorrhage Beatriz Fernández-Muñoz, Cristina Rosell-Valle, Daniela Ferrari, Julia Alba- Amador, Miguel Ángel Montiel, Rafael Campos-Cuerva, Luis Lopez-Navas, María Muñoz-Escalona, María Martín-López, Daniela Celeste Profico, Manuel Francisco Blanco, Alessandra Giorgetti, Elena González-Muñoz, Javier Márquez-Rivas, Rosario Sanchez-Pernaute Downloaded from https://academic.oup.com/stcltm/article/9/9/1085/6406939 by Universidad de Sevilla user on 05 June 2023 TISSUE-SPECIFIC PROGENITOR AND STEM CELLS Retrieval of germinal zone neural stem cells from the cerebrospinal fluid of premature infants with intraventricular hemorrhage Beatriz Fernández-Muñoz 1,2 | Cristina Rosell-Valle 1 | Daniela Ferrari 3 | Julia Alba-Amador 1 | Miguel  Angel Montiel 1 | Rafael Campos-Cuerva 1,4 | Luis Lopez-Navas 5 | María Muñoz-Escalona 1 | María Martín-López 1,2 | Daniela Celeste Profico 6 | Manuel Francisco Blanco 1 | Alessandra Giorgetti 7 | Elena González-Muñoz 8,9,10 | Javier Márquez-Rivas 2,11 | Rosario Sanchez-Pernaute 5 1 Unidad de Producción y Reprogramación Celular (UPRC), Red Andaluza para el diseño y traslación de Terapias Avanzadas, Sevilla, Spain 2 Grupo de Neurociencia aplicada, Instituto de Biomedicina de Sevilla, Sevilla, Spain 3 Department of Biotechnology and Biosciences, University Milan-Bicocca, Milan, Italy 4 Centro de Transfusiones, Tejidos y Células de Sevilla (CTTS), Sevilla, Spain 5 Departamento de Preclínica, Red Andaluza de Diseño y Traslación de Terapias Avanzadas, Sevilla, Spain 6 Fondazione IRCCS Casa Sollievo della Sofferenza, Production Unit of Advanced Therapies (UPTA), San Giovanni Rotondo, Italy 7 Regenerative Medicine Program, Bellvitge Biomedical Research Institute (IDIBELL); Program for Translation of Regenerative Medicine in Catalonia (P-CMRC), Barcelona, Spain 8 Department of Cell Biology, Genetics and Physiology, University of Málaga, Málaga, Spain 9 Department of Regenerative Nanomedicine, Andalusian Center for Nanomedicine and Biotechnology-BIONAND, Málaga, Spain 10 Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). Carlos III Health Institute (ISCIII), Spain 11 Neurosurgery Department, Hospital Virgen del Rocío, Sevilla, Spain Abstract Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology. KEYWORDS cerebrospinal fluid, germinal zone, intraventricular hemorrhage, neural stem cell, neurogenesis, premature infant Received: 26 September 2019 Accepted: 29 April 2020 DOI: 10.1002/sctm.19-0323 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, Inc. on behalf of AlphaMed Press STEM CELLS Transl Med. 2020;9:1085–1101. wileyonlinelibrary.com/journal/sct3 1085 Downloaded from https://academic.oup.com/stcltm/article/9/9/1085/6406939 by Universidad de Sevilla user on 05 June 2023 Correspondence Beatriz Fernández-Muñoz, PhD, Unidad de Producción y Reprogramación Celular (UPRC) C/Max Planck, 3. Edificio Iris 1. 41092, Sevilla, Spain. Email: beatriz.fernandez.munoz@ juntadeandalucia.es Present address María Muñoz-Escalona, Centre for Genomics and Oncological Research (GENYO), Granada, Spain 1|INTRODUCTION Intraventricular hemorrhage (IVH) is a common complication of premature infants, occurring in 15% to 40% of preterm infants weighing less than 1500 g at birth and being particularly common in extremely low birthweight neonates. 1-3 IVH is classified into four grades according to the extent of hemorrhage, development of subsequent ventricular dilatation, and parenchymal involvement: grade I—a hemorrhage restricted to subependymal region; grade II—a hemorrhage bleeding into de ventricles without dilation; grade III—an IVH with ventricular dilatation; and grade IV—an IVH with associated adjacent brain parenchyma infarction. 4 Some authors classify grade IV IVH separately because the presence of periventricular hemorrhagic infarction or other parenchymal lesions is generally not caused simply by extension of IVH into brain parenchyma and should thus be considered as a different pathological condition. 5 Around 10% to 15% of preterm infants develop severe (grade III-IV) IVH and those infants are at high risk to develop posthemorrhagic hydrocephalus, an expansion of the ventricles due to cerebrospinal fluid (CSF) accumulation (reviewed in Reference 6), and to present long-term neurological deficits with cognitive and motor disabilities. 6,7 IVH initiates in the periventricular germinal zone (Gz), also known as germinal matrix, a highly proliferative, highly vascularized region around the lateral ventricles with a dense and fragile, endothelial-lined, vessel network. 8 From 24 to 32 weeks of gestation the Gz is most prominent in the caudo-thalamic groove, forming the ganglionic eminences of the ventricular zone (VZ), where late migrating cortical and thalamic neurons and oligodendrocyte precursors are born. 9 The ultimate cause of Gz bleeding remains unclear, but it is commonly accepted that it results from the combination of Gz vasculature vulnerability and blood pressure fluctuations associated with prematurity. 8 In the hemorrhage phase, there is a rupture of the Gz, occurring most often at the level of the medial ganglionic eminence, that entails loss of neural stem cells (NSCs) and disturbs the cytoarchitecture of the zone leading to abnormal neuronal, ependymal, and glio-genesis. 10,11 Current treatments for IVH are intended to decrease the intracranial pressure that can cause periventricular white matter compression and damage, impairment of brain development, and even death. 12,13 There is a standardized protocol neither for the type nor for the timing of the intervention, 14-16 but it has recently been shown that early removal of hemorrhagic CSF by neuroendoscopic lavage is a safe procedure that may mitigate the adverse effects of the accumulation of blood products, decrease the need for permanent shunt placement, and potentially reduce neurological disability. 17-19 NSCs are the self-renewing, multipotent cells that generate neuronal and glial cell populations during development. During brain development, primary NSCs located in the VZ have radial processes (apical radial glial cells), divide symmetrically, are highly polarized, and express prominin-1 (CD133). 20 Radial glia are more multipotent than the intermediate progenitors of the subventricular zone (SVZ) that include basal radial glia, transient amplifying, and neural progenitors. 21,22 Regional differences in the transcriptional profile of radial glia dictate the fate of their postmitotic progeny. In addition to their differentiation potential, NSCs produce neurotrophic and neuroprotective molecules, making them attractive for regenerative approaches. 23,24 In this regard, clinical-grade human NSC lines are usually obtained from the fetal central nervous system (CNS). Human NSCs can also be isolated from the adult CNS in patients undergoing surgical procedures, 25 or be derived from pluripotent stem cells (PSC) and from somatic cells through reprogramming protocols. 24,26 Advanced therapy medicinal products based on clinical-grade human allogenic fetal NSCs are being tested in clinical trials for various neurological disorders and, although efficacy has yet to be ascertained in a clinical setting, their safety profile has been repeatedly confirmed (reviewed in Reference 24). Significance statement Intraventricular hemorrhage (IVH), occurring in 15% to 40% of preterm births, is frequently associated with long-term neurological deficits. The rupture of the proliferative germinal zone in IVH disturbs late neuronal, ependymal, and glio-genesis. Using a minimally invasive neuroendoscopic procedure, neural stem cells can be retrieved from the cerebrospinal fluid, which can be expanded, cryopreserved, and differentiated in vitro and in vivo, and are not tumorigenic. These cells display distinct hallmarks related to their origin in the germinal zone of the ventral forebrain and could be useful for the development of an autologous cell therapy aiming to attenuate neurocognitive sequelae. 1086 FERNÁNDEZ-MUÑOZ ET AL. Downloaded from https://academic.oup.com/stcltm/article/9/9/1085/6406939 by Universidad de Sevilla user on 05 June 2023 Here we demonstrate that a novel class of NSCs can be robustly isolated from the hemorrhagic CSF of preterm neonates during neuroendoscopic lavage. These NSCs that we named Gz-NCS display distinctive features corresponding to their origin in the ventral forebrain. Gz-NSCs could be useful to develop an autologous cell therapy aiming to reduce neurological disability in preterm infants and to further our understanding of human Gz biology. 2|MATERIALS AND METHODS 2.1 |Hemorrhagic CSF collection CSF samples were obtained from eight preterm infants with grade IV IVH (Table 1) at the Hospital Universitario Virgen del Rocío (Sevilla, Spain). The study was approved by the Hospital Universitario Virgen del Rocío ethical committee and has been performed in accordance with the 1964 Declaration of Helsinki. All samples were obtained after parental informed consent. The SSPA Biobank has coordinated the collection, processing, management, and assignment of the biological samples used in this study, according to the standard operating procedures established for this purpose. Under intraoperative ultrasound guidance 27 (Video S1) the ventricle with the larger amount of blood was punctured with the surgical endoscope (MINOPModular Neuroendoscopy system, Aesculap, Inc., Center Valley, Pennsylvania) and the content was collected. Briefly, the neurosurgical endoscopy system has two irrigation channels, a scope channel, and a working channel, allowing the simultaneous irrigation and collection of liquids during the surgical procedure. The endoscope is introduced through a minimal precoronal corticotomy, one channel is connected to an infusion line, and another channel to an outflow line open at the third ventricle level so that there is no positive resistance to liquid exit, being the system pressure close to 0 H 2 O cm; the infusion rate is determined by gravity (1.8 m) so that there is no risk of inadvertently causing hypertension during the procedure. Additionally, the neurosurgeon constantly monitors the fontanel tension and can open or close the lines to stabilize the pressures. The CSF is collected in 50 mL syringes connected to the outflow channel to minimize exerting an excessive suction. Once the initial hypertension is corrected, continuous irrigation is established using warm, lactated Ringer solution (Baxter, # FE2303) by passive inflow and outflow through the second channel (1.4 mm wide), which was also collected (lavage) in successive syringes until clear. Irrigation was stopped once the fluid within the ventricular system was clear, or at any time if hemodynamic instability appeared. Typically, 1000 to 2000 mL of Ringer solution were used and collected in 50 mL sterile syringes that were immediately closed to maintain sterility. The bleeding area was sealed with fibrinogen beads (Floseal, Baxter #ADS201845). 2.2 |NSC isolation from hemorrhagic CSF CSF samples were transferred to appropriate tubes and centrifuged at 370gfor 10 minutes. The cell pellet was resuspended in N2/B27 medium: Dulbecco's modified Eagle medium (DMEM)-F12 (ThermoFisher Scientific #11530566), 0.1 mM nonessential amino acids (Sigma-Aldrich #RNBG4911), 100 IU penicillin/100 μg/mL streptomycin (Sigma-Aldrich #P0781), 2 μg/mL heparin (Rovi #641647), 1% N2 (ThermoFisher Scientific #11520536), 1X B27 (ThermoFisher Scientific #11530536), 20 ng/ mL FGF (Miltenyi Biotec #130-093-564), 20 ng/mL EGF (Peprotech #AF- 100-15), and 10 ng/mL LIF (Miltenyi Biotec #130-108-156). The cell suspension was seeded onto 20 μg/mL poly-L-ornithine (Sigma-Aldrich # P4957)/20 μg/mL laminin from human placenta (Sigma-Aldrich #L6274) (POL) or Matrigel (Corning #354277)-coated plates. Medium was changed 24-48 hours after seeding. Cells were seeded for expansion at 1 ×10 5 cells/mL in low binding flasks or at 12 000 cells/cm 2 in Matrigel- or POL- coated plates. Matrigel-coated flasks were prepared by incubation with Matrigel diluted in cold DMEM-F12 for 1 hour at room temperature according to manufacturer's instructions. For POL coating, flasks were incubated with 20 μg/mL poly-L-ornithine for 1 hour at 37Corovernight at 4C. Flasks were washed twice with distilled water and they were then further incubated with 20 μg/mL laminin for 2 hours at 37C. Flasks were washed three times with phosphate buffered saline (PBS, ThermoFisher Scientific #A12856-01) before cell seeding. Cells were expanded for 3 (early) and 7-10 (late) passages for characterization. Passage 7, which corresponds to 13 ± 1 accumulated population doublings, was considered “late passage”given that it will not be possible to extensively expand the cells in a clinical setting. Magnetic activated separation (MACS) was performed using the CD133 MicroBead kit (Miltenyi Biotec #130-097-049) following manufacturer's instructions. 2.3 |Immunofluorescence Cells grown over Matrigel-coated coverslips were fixed with 4% paraformaldehyde (SantaCruz Biotechnology #SC-281692), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich #T8787), blocked in PBS (ThermoFisher Scientific #A12856-01) with 1% bovine serum albumin (BSA, Sigma-Aldrich #A8806) for 30 minutes at 37C and incubated with the primary antibody overnight at 4C. Cells were subsequently TABLE 1 Hemorrhagic cerebrospinal fluid samples Batch Weight (g) Sex EGA (weeks) Case 1 2150 Male 33 Case 2 1950 Male 36 Case 3 1600 Male 30 Case 4 1142 Female 27 Case 5 a 3120 Female 42 Case 6 1120 Male 29 Case 7 850 Male 29 Case 8 805 Female 28 a CD133 expression dropped drastically with passages and therefore cells isolated from this sample were excluded from further analysis. Abbreviation: EGA, estimated gestational age at CSF collection. HUMAN VENTRAL FOREBRAIN NEURAL STEM CELLS 1087 Downloaded from https://academic.oup.com/stcltm/article/9/9/1085/6406939 by Universidad de Sevilla user on 05 June 2023 incubated with the secondary antibody for 30 minutes at 37C and mounted with ProLong Gold Antifade Mountant with 40,6-diamidino- 2-phenylindole (DAPI, ThermoFisher Scientific #P36930). Primary and secondary antibodies are listed in Table S1. For Ki-67 detection, we first performed an antigen retrieval step in which cells were heated for 10 seconds in a microwave with citrate buffer pH 6.0 (Sigma- Aldrich #C9999) letting cells cool down 20 minutes. Acquisition of fluorescence images was performed in a Leica TCS-SP5 or a Nikon Eclipse Ti fluorescence microscope. Images were processed using the Adobe Photoshop CS5 or ImageJ software. 28 Positive cells were counted using the ImageJ software from at least three random fields per preparation. 2.4 |Flow cytometry For CD133, CD24, CD34, CD45, PODXL, IL1RAP, and MHC detection, live cells were blocked in PBS with 1% BSA and incubated with conjugated antibodies for 15 minutes at 4C. For TREK2, FZD5, and DLK1 analysis, cells were fixed with 3.7% formaldehyde (Sigma- Aldrich #F8775), permeabilized with 0.1% Triton X-100 (Sigma- Aldrich #T8787), blocked in PBS with 1% BSA and incubated with the primary antibody for 30 minutes at 4C. Cells were subsequently incubated with the secondary antibody for 30 minutes at 4C. Antibodies are listed in Table S1. Fluorescence was estimated with a Macs Quant flow cytometer (Miltenyi Biotec) and results were analyzed with the MacsQuantify 2.10 and FloJ v10 software. Appropriate isotype controls were run in parallel with the samples. Gating strategies for the different antibody panels are shown in Figure S1. 2.5 |Transcriptomic analysis 2.5.1 |Control stem cell samples Human fetal NSCs (n = 4) were derived from the forebrain of 15- to 22-week-old fetuses that had undergone spontaneous in utero death (miscarriage). Tissue procurement was approved by the Ethics Committee of the Institute “Casa Sollievo della Sofferenza”after receiving the mother's informed, written consent. These fetal NSC lines have been extensively characterized. 29-31 PSC-derived NSCs (iPS-NSC, n = 6) were differentiated from embryoid bodies (EBs) in TeSR2 medium (Stemcell Technologies #05860) spiked with Rock inhibitor (Y-7632; 10 μM; Tocris Bioscience #1254). After 7 days, EBs were plated on Matrigel and cultured in neural differentiation media. On day 10, 0.1 μMretinoic acid (RA, Sigma-Aldrich #R 2625) was added to the medium. On day 15, neural tube-like rosettes were mechanically detached and FIGURE 1 Neuroendoscopy and CSF collection. A, Schematic representation of the germinal zone localization (in blue) around the ventricles (axial view) that comprises the ventricular and subventricular zones where neural stem cells are found (box). B, Computed tomography axial brain images depicting the bleeding area close to the head of the caudate nucleus and the presence of blood inside the ventricular system (arrows) in one of the cases. C, Recovery of hemorrhagic CSF and irrigation fluid from preterm infants with IVH grade IV. Images of the surgical intervention by neuroendoscopy: preparation (i); neuroendoscopic imaging of bleeding area before (ii) and after (iii) irrigation, and, before (iv) and after (v) sealing; collection of irrigation fluid (vi) 1088 FERNÁNDEZ-MUÑOZ ET AL. Downloaded from https://academic.oup.com/stcltm/article/9/9/1085/6406939 by Universidad de Sevilla user on 05 June 2023 FIGURE 2 Legend on next page. HUMAN VENTRAL FOREBRAIN NEURAL STEM CELLS 1089 Downloaded from https://academic.oup.com/stcltm/article/9/9/1085/6406939 by Universidad de Sevilla user on 05 June 2023 cultured in neural differentiation media with 20 ng/mL FGF (Miltenyi Biotec #130-093-564) and 20 ng/mL EGF (Peprotech #AF-100-15). Cells were expanded in suspension as neurospheres or in adhesion over Matrigel during 6 to 7 passages before RNA extraction for transcriptomic analysis. Three human PSC lines were included for reference. CBiPS were derived from CD133 + umbilical cord cells. These cells are available from the Spanish national repository, (BNLC) and the data regarding cell characterization can be downloaded at http://www.eng.isciii.es. WA09 (H9) were purchased from WiCell and the characterization data can be downloaded at www.wicell.org. L6-iPS was generated by our group (see experimental procedures in Supplementary Information) and we are in the process of banking it at the Spanish BNLC. Characterization of this cell line is provided in Figure S2. Umbilical cord blood samples (CD34 + HSC, n = 3) were obtained from the Banc de Sang i Teixits, Barcelona. CD34 + cell purification was performed as previously described. 32 Briefly, mononuclear cells (MNC) were isolated from CB using Lympholyte-H (Cederlane #CL5015) density gradient centrifugation. CD34 + cells were positively selected using Mini-Macs immunomagnetic separation system (Miltenyi Biotec #130-046-702,). Purification efficiency was determined by flow cytometry analysis staining with CD34-phycoerythrin (PE; Miltenyi Biotec #130-120-520) antibody. 2.5.2 |Expression microarrays RNA was extracted with RNeasy Mini kit (Qiagen #74104) following the instructions of the manufacturer and sent to the Genomics Unit of the Andalusian Center of Molecular Biology and Regenerative Medicine (CABIMER). RNA quality was analyzed by the Bioanalyzer 2100 (Agilent). All samples had RNA Integrity Number (RIN) higher than 9. cDNA was synthetized, labeled with biotin and hybridized with independent Human Clariom-S Microarrays, (Affymetrix #902927) following Affymetrix protocol. Microarrays were scanned with Affymetrix GeneChip Scanner 3000 7G, and the obtained data were analyzed with the Affymetrix GeneChip Command Console 2.0 software. The microarray expression data set is publicly available at the GEO repository under the identifier GSE124361. Further analyses were performed using the Transcriptome Analysis Console (TAC, Affymetrix) v4.0 software and R version 3.5.0. 33 Functional enrichment analysis was performed using the bioinformatics tool EnrichR (http://amp.pharm.mssm.edu/Enrichr/). 34,35 Neuroanatomical references were obtained from the Allen Atlas of the developing human brain (www.brainspan.org). 36 2.5.3 |RT-PCR First, 0.1 μg of RNA were used for cDNA synthesis using Oligo-dT (Invitrogen #18418012), RNase OUT (Invitrogen #10777-014) and SuperScriptII Retrotranscriptase (Invitrogen #18064-014). PCR products were obtained using 5 ng of cDNA and Mytaq Red DNA Polymerase (Bioline #BIO-21108) following the manufacturer's instructions. Oligonucleotides used for amplification are provided in Table S2. 2.6 |Transplantation into nude mice Animal care and experimental procedures were conducted according to the current National and International Animal Ethics Guidelines and approved by the Italian Ministry of Health. Ten 7- to 8-week-old female athymic Nude-Foxn1nu mice (#6902F, Envigo) received a single injection of 300 000 CD133+ cells in the striatum. Stereotaxic coordinates were determined using the Paxinos and Franklin atlas 37 and the injections were done at: anteroposterior, 0 mm; dorsolateral, 2.5 mm from bregma; and dorsoventral, −2.7 mm from dura matter. All the injections were performed on the right hemisphere. Cells were injected in 3 μLduring 5 minutes using HBSS (Gibco #14175095) as vehicle. Three animals were sacrificed at 3 weeks to assess survival and proliferation and seven animals were sacrificed at 6 months. Transplantation experiments and analyses were performed as previously described for fetal NSCs. 29,38 For immunohistochemical analysis, brains were perfused-fixed with ice-cold 4% paraformaldehyde (Sigma #158127), postfixed overnight, cryoprotected and sectioned in a cryostat. Twenty micrometers coronal sections were serially collected and processed for immunofluorescence. Quantification of transplanted cells was done using Image J cell counter-plugin on images acquired in a Nikon C2 confocal microscope and the NIS Elements 1.49 software. All cells expressing human nuclear antigen (huN) were counted in sections spanning the graft area, and the total number was calculated using Abercrombie correction 39 and expressed as the percentage over injected cells. Migration was estimated as the distance between the first and last sections containing huN FIGURE 2 Isolation of NSC-like cells from the CSF of IVH patients. A, Phase-contrast microphotographs of CSF-derived NSC cultures at different days in vitro (DIV) after isolation. Scale bar: 100 μm. B, Exponential growth kinetics of 3 representative NSC lines grown on Matrigel. C, Phase-contrast microphotographs of cells at early (0) and late (10) passages grown on matrigel. D, Proliferation was assessed by quantification of Ki-67 expression at early (3) and late (7) passages. A representative confocal section is shown. Scale bar 25 μm. E, Flow cytometry analysis of CD133, CD24, CD34 and CD45 at early (3) and late (7) passages. There were no significant differences between conditions. Data are shown as mean ± SEM of 5-7 independent biological samples. The 42-week-old case (pink symbols) was excluded from further analysis. F, Co-expression of CD133 with CD24 and CD34 at early and late passages. G, Representative microphotographs of the NSC-like cells obtained from the CSF and the lavage fluid at 13 days after isolation. Scale bar: 100 μm. H, CD133 analysis by flow cytometry of the NSC-like cells obtained from the CSF and the lavage fluid at passage 3. *P< .05 1090 FERNÁNDEZ-MUÑOZ ET AL. Downloaded from https://academic.oup.com/stcltm/article/9/9/1085/6406939 by Universidad de Sevilla user on 05 June 2023 FIGURE 3 Legend on next page. HUMAN VENTRAL FOREBRAIN NEURAL STEM CELLS 1091 Downloaded from https://academic.oup.com/stcltm/article/9/9/1085/6406939 by Universidad de Sevilla user on 05 June 2023 positive cells. Coexpression of Ki67, glial fibrillary acidic protein (GFAP), and OLIG2 was counted in three serial sections of the transplanted animals (n = 3-7) and expressed as percentage over total huN + cells in those sections. 2.7 |Statistics Data are presented as mean ± SEM. Significance was determined using two-tailed Student's ttest for comparisons between two samples. P< .05 was considered significant. Paired t-tests or repeated measures (R) ANOVA were used to compare samples from the same individual at different stages. All statistical analyses were performed using the GraphPad Prism 8.01 software. Bioinformatic analyses were performed using the Affymetrix and R software, using t, ANOVA, and R-ANOVA tests and selected thresholds, as indicated in the text and figure legends. A false discovery rate (FDR) <5% was established for significance. 3|RESULTS 3.1 |Hemorrhagic CSF of preterm IVH patients contains NSCs Eight consecutive cases with a clinical and radiological diagnosis of grade IV IVH (Table 1) underwent a ventricular neuroendoscopy to seal the bleeding area and remove the hemorrhagic CSF from the ventricular cavities (Figure 1, Video S1). Neuroendoscopic lavage was performed following the technique reported by Schulz et al 18 with a few modifications as detailed in the Methods section. The content of the first syringe (collected before starting the irrigation) was processed for cell recovery. After centrifugation, the cell pellet was initially seeded on POL or Matrigel-coated plates and cultured in an N2/B27 serumfree medium with mitogens; 24-48 hours after seeding, small aggregates were observed amidst abundant erythrocytes and blood cells in suspension (Figure 2A). Cells were enzymatically dissociated and passaged as neurospheres or in adhesion for 10 passages showing a doubling time of 3.94 ± 0.33 days and maintaining a stable growth curve and morphology (Figure 2B,C). Quantification of Ki-67 showed no significant decrease in proliferation between early and late passages (12.77 ± 8.88 vs 18.83 ± 7.44; Figure 2D). Likewise, expression of the stem cell marker prominin-1 (CD133) was maintained through passages (58.99 ± 8.7 vs 57.93 ± 10.82; Figure 2E). An exception was the 42-weeks-old sample (Figure 2E, pink symbols) in which the percentage of CD133 + cells dropped drastically upon passaging. This case was excluded from further analyses given that IVH in full-term neonates most often originates in the choroid plexus. 8 We next analyzed whether the cell population obtained from hemorrhagic CSF had a similar expression pattern of CD surface antigens than that described for fetal NSCs. 40,41 Like fetal NSCs, most cells in CSF samples were positive for CD133 and all were negative for CD45, displaying a variable expression of CD24 (Figure 2E and Table S3). Intriguingly, some samples contained a substantial percentage of CD34 positive cells (Figure 2E and Table S3), which is not expressed by fetal forebrain NSCs. 40 On average, 18.10 ± 7.65% of the cells were CD133 + CD34 + at passage 7, and 49.42 ± 10.65 of the cells were CD133 + CD24 + at passage 7. Despite no significant differences, there was a trend to an increase of CD24 double positive cells with passages (29.13 ± 7.4 vs 49.42 ± 10.65; Figure 2F and Table S3). We attempted to recover cells from subsequent CSF samples, collected once the irrigation started, but the flow cytometry analyses showed fewer CD133 + cells in the lavage fluid (66.26 ± 12.47 vs 26.18 ± 15.83; P= .02 [paired ttest]; Figure 2G,H) so the rest of the study was carried out using only the first tube of hemorrhagic CSF from each case. In another experiment, we failed to recover NSC-like cells from nonhemorrhagic CSF samples (see supplementary experimental procedures and Figure S3) obtained from a different set of patients with obstructive hydrocephalus, although in some cases, changing to a serum-based media allowed us to grow fibroblast-like, adherent cells from samples with large CSF volumes (>20 mL). 3.2 |NSCs isolated from the hemorrhagic cerebrospinal fluid present distinctive regional hallmarks We next performed a transcriptomic analysis to study the differences and similarities between NSCs isolated from hemorrhagic CSF, fetal forebrain NSCs and NSCs derived from iPSCs. Given that CSF samples contained mostly blood cells we also included in the analysis FIGURE 3 NSC cells isolated from the CSF display a ventral forebrain gene-expression profile. A, PCA analysis of global gene-expression profiles. B, Venn diagrams showing the number of differentially expressed genes (DEG), 2-fold change, FDR P< .05. C, Volcano plot of DEG in NSCs from fetal brain (red) and CSF (blue) sources. Highlighted are markers that identify regional populations including genes that have been previously associated with germinal zones and forebrain regionalization (see also schematic in D) and putative candidates for prospective identification of germinal zone NSCs. Expression levels of NSC and regional forebrain markers (D) and candidate DEG genes that could identify this NSC population (E). F, Semi-quantitative RT-PCR of NSC markers and candidate DEG genes. G, Enrichment network analysis of upregulated genes relative to fetal NSCs, profiled across brain regions according to the Allen brain atlas, and schematic neuroanatomical representation on coronal brain sections showing their periventricular location. A, anterior; BNST, bed nuclei of the stria terminalis; cGE, caudal ganglionic eminence; Cd, caudate nucleus; D, dorsal; DThal, dorsal thalamus; GP, globus pallidus; lGE, lateral ganglionic eminence; mGE, medial ganglionic eminence; P, posterior; Put, putamen; Sept, septum; SVZ, subventricular zone; Thal, thalamus; V, ventral; V Pall, ventral pallidum; VZ, ventricular zone 1092 FERNÁNDEZ-MUÑOZ ET AL. Downloaded from https://academic.oup.com/stcltm/article/9/9/1085/6406939 by Universidad de Sevilla user on 05 June 2023 5|CONCLUSION Here we describe a novel class of NSCs, the Gz-NSCs that can be easily and robustly isolated from the CSF of preterm neonates with grade IV IVH undergoing neuroendoscopic lavage. We found that these cells, while being similar to fetal forebrain NSCs, have several distinctive hallmarks related to their regional and developmental origin in the ventral forebrain. Gz-NSCs can be expanded and cryopreserved showing in vitro and in vivo differentiation potential, and pose no ethical concerns as the fluid is usually discarded. Thus, Gz-NSCs could represent an optimal source for the development of an autologous cell therapy for infants with IVH, as well as a useful tool for studying the late stages of human neural development. ACKNOWLEDGMENTS Authors are grateful to Mónica Perez Alegre, Eloisa Andújar and Paloma Dominguez for technical assistance with the experiments performed in the Genomic and Confocal Units of the Andalusian Center of Molecular Biology and Regenerative Medicine (CABIMER) and to Gloria Carmona and all the members of the Unidad de Producción y Reprogramación Celular (UPRC) for technical help and support. This work was supported by research funds from the Andalusian Consejería de Salud to the Red Andaluza de Diseño y Traslación de Terapias Avanzadas with contribution from the COST Action CA16122 for STSM and networking. AG is supported by Ramon y Cajal Program (RyC-2013-13221), MINECO(SAF2016-80205-R) and CERCA Program/Generalitat de Catalunya. CONFLICT OF INTEREST B.F.M., E.G.M., R.S.P., and J.M. are authors of a patent application for the use of CSF-NSCs (nº application European Patent Office: 200930943). The other authors indicated no potential conflicts of interest. AUTHOR CONTRIBUTIONS B.F.-M.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; C.R.-V.: collection and assembly of data, data analysis and interpretation; D.F.: collection of data, data analysis and interpretation; J.A.-A., M. A.M., R.C.-C., M.M.-L., D.C.P., M.F.B., A.G.: collection of data; L.L.-N.: assembly and analysis of data, administrative support; E.G.-M.: conception and design, collection of data; J.M.-R.: conception and design, provision of study material or patients, collection of data, data analysis and interpretation; R.S.-P.: conception and design, financial support, assembly of data, data analysis and interpretation; All authors: revision and final approval of manuscript. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available upon reasonable request to the corresponding author. 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STEM CELLS TRANSLATIONAL MEDICINE. 2018;7:847-856. SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section at the end of this article. How to cite this article: Fernández-Muñoz B, Rosell-Valle C, Ferrari D, et al. Retrieval of germinal zone neural stem cells from the cerebrospinal fluid of premature infants with intraventricular hemorrhage. STEM CELLS Transl Med. 2020;9: 1085–1101. https://doi.org/10.1002/sctm.19-0323 HUMAN VENTRAL FOREBRAIN NEURAL STEM CELLS 1101 Downloaded from https://academic.oup.com/stcltm/article/9/9/1085/6406939 by Universidad de Sevilla user on 05 June 2023