Patent Application
20210207089
The present invention provides a novel method to isolate and expand neural stem cells (NSCs) from cerebrospinal fluid (CSF) of premature babies with Intraventricular haemorrhage, which produces a population enriched in CSF-NSC cells free of contaminating fibroblasts and other cell types. The present invention also includes substantially pure populations of CSF-NSC cells, and their use to treat and prevent diseases and injuries, including Intraventricular haemorrhage and post-hemorrhage hydrocephalus.
Intraventricular haemorrhage (IVH) is a common cause of morbidity and mortality in premature infants. The incidence of premature infants with IVH has declined in recent years, but remains a significant problem in infants with very low birth weight (VLBW<1500 g) and extremely low birth weight (ELBW<1000 g). IVH is classified according to the degree of haemorrhage and subsequent ventricular dilatation (Grade I-II as defined moderate IVH, and grade III-IV as defined severe IVH) (Premature infants with severe IVH present higher risk to develop post-hemorrhage hydrocephalus (PHH) or periventricular leukomalacy, and exhibit long-term neurological deficits with cognitive and psychomotor disabilities. No cure for IVH has been developed so far.
Typically, IVH initiates in the subependymal germinal matrix, the source of cerebral neural stem cells during cortex development, between approximately the 10th and 24th gestational weeks (Ballabh, 2010. Pediatr. Res. 67, 1-8). In the haemorrhage stage, there is a rupture of the germinal matrix that entails loss of neural stem cells and disturbs the normal cytoarchitecture of the ventricular zone compromising the organization and function of the cerebral cortex (reviewed in Guerra, 2014. Fluids Barriers CNS 11, 1-10).
Neural stem cells (NSCs) have differentiation and self-renewing potential and express neuroprotective factors, capabilities that make them suitable to regenerate lost tissue having a great therapeutic potential for the treatment of different pathologies (Ludwig, 2018. Neural Regen. Res. 13, 7-18; Tang, 2017 Cell Death Dis. 8). This potential has been tested in preclinical studies showing some success in a variety of animal models of different nervous system diseases and in clinical trials for spinal cord injury, amyotrophic lateral sclerosis, glioma, cerebral palsy and other neurological disorders. Although data from many of these clinical trials are still being compiled, some improvements in neurological function has been reported and safety of the NSC-based therapies has been confirmed (reviewed in Tang, 2017). NSCs can be isolated from the central nervous system (CNS) of fetuses and adult tissue, but these procedures require human embryos or invasive procedures, respectively, which have obvious limitations. NSCs can be also derived from pluripotent stem cells and somatic cells through reprogramming protocols (Tang, 2017) but these are poorly standardized procedures and show low efficacy giving rise to a low purity NSCs population. Cerebrospinal fluid of spina bifida cases has been also recently proposed as a new source for NSC/NPCs. Nonetheless, NSCs from the CNS of fetuses remain the most used cell type for clinical use. Indeed, several companies are using this cell type in clinical trials for neurological disorders (reviewed in Tang, 2017). Despite the encouraging results of some of these clinical trials, the scarcity of source material and the ethical problems associated with isolation of the NSC is an obvious constraint for the use of these cells as a therapy to improve patient quality of life.
Here we demonstrate that a novel type of neural stem cells can be easily and robustly isolated from preterm infants with IVH. We have characterized the cell population obtained from the cerebrospinal fluid (CSF) and found that these cells are very similar to foetal forebrain NSCs, and not to other stem cell types, such as CD34 positive cord blood or bone marrow mesenchymal stem cells. However, these CSF-NSCs present several distinctive hallmarks such as ventral regional transcription factors and an increased expression of podocalyxin (PODXL) or IL1RAP. These CSF-NSCs are directly isolated from the liquid obtained from the cerebral ventricles during neuroendoscopic irrigation performed as treatment of posthemorragic hydrocephalus, and pose no ethical concerns as the fluid is usually discarded. CSF-NSCs could be useful for the development of autologous therapies for infants with IVH and PHH or perhaps for developing allogeneic therapies for different neurological disorders, and for furthering our understanding of human late brain development.
Table 1. —Antibodies.
Table 2. —Identification of tissue transcriptional profiles corresponding to genes upregulated in CSF-NSCs with respect to fetal NSCs. Shown are the 5 top categories with the number of genes, percentages of the differentially expressed genes, significance and the list of genes. Note that genes can appear in several lists and some genes may not map to any of the archived tissues (EnRichR-ARCHS4 Tissue).
Table 3. —Identification of tissue transcriptional profiles corresponding to genes upregulated in fetal NSCs with respect to CSF-NSCs. FDR=0.01 N=3 biological samples at P3 and P7. Shown are the 5 top categories with the number of genes, percentages of the differentially expressed genes, significance and the list of genes. Note that genes can appear in several lists and some genes may not map to any of the archived tissues (EnRichR-ARCHS4 Tissue).
Table 4. Identification of tissue transcriptional profiles corresponding to genes differentially expressed in CSF-NSCs with respect to fetal NSCs. FDR=0.05 N=5 biological samples at P3 (EnRichR-ARCHS4 Tissue).
Table 5. Antibodies.
Table 6. Primers.
In the present invention, we demonstrate that premature babies with IVH are shedding NSCs from the germinal matrix into the CSF, a material that is regularly removed from these patients to ameliorate effects related to intracranial pressure and that is usually discarded in most of the hospitals. In this regard, the authors obtained from one batch approximately 3×106 cells at passage 0. After 4 passages, they generated 250×106 cells, a number we estimated enough for an autologous treatment, at least to put back in the germinal zone the NSCs that infants lose during IVH. In addition, we have demonstrated here that CSF-NSCs are phenotypically stable through passages and they adequately proliferate maintaining a stable karyotype, indicating that a large amount of stable cells can be obtained safely. Also, the transcriptomic profile suggested that GM-NSC were less committed than fetal NSC, which were derived from fetuses at earlier developmental stages (15-22 for fetal vs 26-36 weeks EGA in IVH). This is most likely related to a larger contribution of the cortical SVZ, which is greatly expanded in humans, than the VZ, to fetal dorsal forebrain volume (see schematic in
The expression arrays of stem cells obtained from CSF samples show that gene-expression profile is closer to foetal NSCs than to IPS-derived neural stem cells, bone marrow mesenchymal or cord blood hematopoietic stem cells. However, at FDR<0.05 and 2-fold change there are over 1000 genes differentially expressed in CSF-NSCs. Among those markers relatively overexpressed in the GM-NSC, FZDS, HLA related markers, Podocalyxin and IL1RAP are membrane-bound proteins that can be useful to isolate these cells.
We also show in this invention that CSF-NSCs cannot be obtained from non-hemorrhagic CSF. However, fibroblast-like cells can be isolated from these samples meaning that non-haemorrhagic CSF is a new source for fibroblast/mesenchymal stem cells isolation.
As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated: A “stem cell” refers to an undifferentiated, multipotent, self-renewing, cell. A stem cell is able to divide and, under appropriate conditions, has self-renewal capability and can include in its progeny daughter cells that can terminally differentiate into any of a variety of different cell types. A stem cell is capable of self-maintenance, meaning that with each cell division, one daughter cell will also be on average a stem cell.
The non-stem cell progeny of a stem cell is typically referred to as “progenitor” cells, which are capable of giving rise to various cell types within one or more lineages. The term “progenitor cell” refers to an undifferentiated cell derived from a stem cell, and is not itself a stem cell. Some progenitor cells can produce progeny that are capable of differentiating into more than one cell type. A distinguishing feature of a progenitor cell is that, unlike a stem cell, it does not exhibit self-maintenance, and typically is thought to be committed to a particular path of differentiation and will, under appropriate conditions, eventually differentiate along this pathway.
The term “precursor cells” refers to the progeny of stem cells, and thus includes both progenitor cells and daughter stem cells.
Stem cells and progenitor cells derived from a particular tissue are referred to herein by reference to the tissue from which they were obtained. For example, stem cells and progenitor cells obtained from cerebrospinal fluid (CSF) of premature babies with Intraventricular haemorrhage are referred to as “NSC-CSF” or “CSF-NSCs” or germinal matrix-NSC.
A “clonogenic population” refers to a population of cells derived from the same stem cell. A clonogenic population may include stem cells, progenitor cells, precursor cells, and differentiated cells, or any combination thereof.
The term “purified” or “enriched” indicates that the cells are removed from their normal tissue environment and are present at a higher concentration as compared to the normal tissue environment. Accordingly, a “purified” or “enriched” cell population may further include cell types in addition to stem cells and progenitor cells and may include additional tissue components, and the term “purified” or “enriched” does not necessarily indicate the presence of only stem cells and progenitor cells.
The present invention thus provides populations of cells enriched in stem cells obtained from cerebrospinal fluid (CSF) of premature babies with Intraventricular haemorrhage, which are preferably substantially free of contaminating fibroblasts and other cells. More particularly, these cells are preferably obtained from the ventricle of premature babies with the larger amount of hematoma, said ventricle is punctured with the surgical endoscope under intraoperative ultrasound guidance. When ventricular cavities are approached, under direct vision, continuous irrigation is established using warm lactate-free Ringer solution, by passive inflow via an infusion system through the irrigation channel of the endoscope. Simultaneously, a passive outflow is ensured through a second channel to balance the intracranial volume and avoid any significant changes in intracranial pressure. This outflow is preferably collected for subsequent recovery of stem cells through a three-way connection attached to syringes with preferably a luer-lock connection in order to assure sterility and minimal handling of haemorrhagic CSF. Irrigation is stopped once the fluid within the ventricular system is clear or hemodynamic instability appears during surgery. Typically, 1000-2000 ml of Ringer solution are used and collected in sterile syringes that are immediately closed with a cap to maintain a sterile liquid (
Preferably, after collection, CSF is centrifuged and a big red cell pellet is obtained. Cell suspension is then seeded in PLO-laminin or matrigel coated plates (see Experimental procedures for details of the protocol). Medium is preferably changed after 24-48 h and some groups of cells in the NSC-size and tissue spheres slightly adhered to the plate should be observed. 3-5 days after isolation some NSCs-like cells coming out from tissue spheres should be clearly detected. Around day 7 after isolation there should be some neurospheres in suspension and cells in adhesion starting to growth. Around day 8 after isolation cells should be passaged as neurospheres with accutase or in adhesion over matrigel or PLO/laminin coated plates (
At any rate, these populations, populations of cells enriched in CSF-NSCs obtained from cerebrospinal fluid (CSF) of premature babies with Intraventricular haemorrhage, are advantageous over previously described populations of purified stem cells and progenitor cells. In addition, these cell populations, preferably, do not include fibroblasts, which lead to undesired scar formation when administered to a wound or disease site. In addition, contaminating cells, such as fibroblasts, can proliferate more rapidly than stem cells and compete with stem cells in repopulating a tissue site when administered therapeutically.
Thus, in various embodiments, an enriched cell population of the present invention comprises at least 40%, 50%, 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% CD133 positive CSF-NSCs obtained from cerebrospinal fluid (CSF) of premature babies with Intraventricular haemorrhage, as indicated by the presence of one or more stem cell markers, such as CD133. In fact, such cell populations of the present invention, CSF-NSCs, are preferably positive for CD133, and optionally for CD34 and CD24, and negative for CD45 (
In certain embodiments, the purified cell populations of the present invention are present within a composition, e.g., a pharmaceutical composition, adapted for and suitable for delivery to a patient, i.e., physiologically compatible. Accordingly, the present invention includes compositions comprising a stem cell population of the present invention and one or more of buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
In related embodiments, the present invention provides a pharmaceutical composition that comprises the purified cell populations provided herein and a biological compatible carrier or excipient, such as 5-azacytidine, cardiogenol C, or ascorbic acid.
In related embodiments, the purified cell populations are present within a composition adapted for or suitable for freezing or storage. For example, the composition may further comprise fetal bovine serum and/or dimethylsulfoxide (DMSO).
The present invention further provides methods of treating or preventing injuries and diseases or other conditions, comprising providing a cell population of the present invention, i.e., a population enriched in stem cells and progenitor cells, to a patient suffering from said injury, disease or condition. In particular embodiments, the cell population was generated using a tissue sample obtained from the patient being treated (i.e., autologous treatment). In other embodiments, the cell population was obtained from a donor, who may be related or unrelated to the patient (i.e., allogeneic treatment). The donor is usually of the same species as the patient, although it is possible that a donor is a different species (i.e., xenogeneic treatment).
In various embodiments, the stem cell populations and related compositions are used to treat a variety of different diseases, including but not limited to inflammatory diseases, demyelinating diseases, mental disorders, neurodegenerative diseases such as ELA, Alzheimer or Parkinson, neuromuscular diseases, and preferably for the treatment, preferably the autologous treatment, of premature babies having or suffering from Intraventricular haemorrhage or post-haemorrhage hydrocephalus.
In specific embodiments, the present invention provide a methods for treating or preventing premature babies having or suffering from Intraventricular haemorrhage or post-haemorrhage hydrocephalus. These methods comprise providing a cell population of the present invention, wherein said cell population is enriched in CSF-NSCs, to a patient diagnosed, suspected of having, or being at risk of Intraventricular haemorrhage or post-haemorrhage hydrocephalus. In a preferred embodiment, these are isolated from the patient being treated.
Cell populations and related compositions of the present invention may be provided to a patient by a variety of different means. In certain embodiments, they are provided locally, e.g., to a site of actual or potential injury or disease. In one embodiment, they are provided using a syringe to inject the compositions at a site of possible or actual injury or disease. In one embodiment, they are administered to the bloodstream intravenously or intra-arterially. The particular route of administration will depend, in large part, upon the location and nature of the disease or injury being treated or prevented.
Accordingly, the invention includes providing a cell population or composition of the invention via any known and available method or route, including but not limited to oral, parenteral, intravenous, intra-arterial, intranasal, intramuscular and intracranial injection or administration. Preferably, a cell population or composition of the invention is administered at caudate nucleus. The development of suitable dosing and treatment regimens for using the cell populations and compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intramuscular and intracranial injection or administration and formulation, will again be driven in large part by the disease or injury being treated or prevented and the route of administration. The determination of suitable dosages and treatment regimens may be readily accomplished based upon information generally known in the art and obtained by a physician.
Treatment may comprise a single treatment or multiple treatments. In particular, for preventive purposes, it is contemplated in certain embodiments that purified cell populations of the invention are administered during or immediately following a stress that might potentially cause injury.
The present invention also provides kits useful in the preparation and/or use of the purified cell populations of the present invention, which are enriched in stem cells. For example, in one embodiment, a kit useful in the preparation of the purified cell populations is provided that comprises an agent that binds a cell surface marker of stem cells or progenitor cells, and conditioned medium. For example, a kit may include: a first container comprising an antibody specific for a stem cell surface marker, wherein said antibody is adapted for isolation or detection, e.g., by being conjugated to a fluorescent marker or magnetic bead; and a second container comprising conditioned medium. In various related embodiments, the kits may further comprise one or more additional reagents useful in the preparation of a cell population of the present invention, such as cell culture medium, and enzymes suitable for tissue processing. The kit may also include instructions regarding its use to purify and expand stem cells obtained from a tissue sample. In other embodiments, the kits may further comprise a means for obtaining a tissue sample from a patient or donor, and/or a container to hold the tissue sample obtained.
The following examples serve to illustrate but they do not limit the present invention.
The study was approved by the Hospital Virgen del Rock) de Sevilla ethical comitee and CSF samples were obtained after parental informed consent. CSF samples were obtained from 27-36 weeks (EGA) preterm infants by neuroendoscopy at the Hospital Universitario Virgen del Rock) (Sevilla). The ventricle with the larger amount of hematoma was punctured with the surgical endoscope (AesculapMlnop™) under intraoperative ultrasound guidance. When ventricular cavities were approached, under direct vision continuous irrigation was established using warm lactate-free Ringer solution, by passive inflow via an infusion system through the irrigation channel of the endoscope. Simultaneously, a passive outflow was ensured through the second channel (1.4 mm wide) to balance the intracranial volume and avoid significant changes in intracranial pressure. This outflow was collected for subsequent recovery of cells through a three-way connection attached to 50 ml syringes with luer-lock connection in order to assure sterility and minimal handling of CSF. Irrigation was stopped once the fluid within the ventricular system was clear or at any time if hemodynamic instability appeared. Typically, 1000-2000 ml of Ringer solution were used and collected in 50 ml sterile syringes that were immediately closed to maintain sterility.
CSF samples were maintained at 4° C. until NSCs isolation. 2-24h after collection CSF was transferred to appropriate tubes and was centrifugated at 370 g for 10 min. Pellet was resuspended in 2 mL and the resulting supernatant after first centrifugation was centrifuged twice again obtaining more pellet. Cell suspension was counted and seeded in poli-L-ornithine Sigma/laminin from human placenta (Sigma) or hESC qualified matrigel (Corning) coated plates in NDMBL medium (DMEM/F12 (Thermo), 0.1 mM non-essential aminoacids (Sigma), 100 IU penicilin/100 μg/mL streptomycin (Sigma), 2 μg/mL heparin (Sigma) 1% N2 (Thermo), 1×B27 (Thermo), 20 ng/mL FGF (Miltenyi), 20 ng/mL EGF (Preprotech), 10 ng/mL LIF (Miltenyi)). Media was changed 24-48 h after seeding. Cells were seeded for expansion at 0.5×106/mL cells in low binding flasks or at 12.000 cells/cm2 in matrigel coated plates. Cells were expanded for 3 (early) and 7-10 (late) passages for characterization. Passage 7 was considered late-passage given that cells cannot be extensively expanded in a clinical setting. CD133 MACS sorting was performed using human CD133 microbeads (Miltenyi Biotec) following manufacturer instructions.
Fibroblasts Isolation from Non-Hemorrhagic CSF
Cells were cultured with a medium base of high glocuse DMEM supplemented with 10% FBS (Sigma), 0.1 mM non-esential aminoacids (Sigma), 100 IU penicilin/100 μg/mL streptomycin (Sigma) and 2 mM Glutamax (Thermo).
Human fetal NSC were derived from the forebrain of 15-22 weeks (EGA) 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. Fetal NSC lines have been extensively characterized (Mazzini et al., 2015 J. Tranl. Med. 13, 17; Vescovi et al., 1999 Exp. Neurol. 156, 71-83; Gelati et al. 2013. Methods Mol Biol. 2013; 1059:65-77).
The iPS lines used were generated in our lab from adult dermal fibroblasts by electroporation with episomal plasmids containing OCT4, SOX2, NANOG, KLF4, LIN28, c-MYC and SV40LT (MSU-EPI-hiPSC) or by transduction with retroviral vectors for the overexpression of OCT4, SOX2 and KLF4 (E1 L6-hiPSC). We also included CBiPS1sv-4F-5 derived from CD133+ umbilical cord cells by infection with sendai virus and embryonic stem cells WA09 (H9) (iPS cell lines, Cell Lines National Bank, http://www.eng.isciii.es).
For neural differentiation, iPS were cultured as embryoid bodies (EB) in TeSR2 medium (Stemcell Technologies) spiked with Rock inhibitor (Y-27632; 10 μM; Tocris Bioscience). After 7 days, EB were plated on matrigel and cultured in neural differentiation media. On day 10, retinoic acid (RA) was added to the medium. On day 15, neural tube-like rosettes were mechanically detached and cultured in neural differentiation media with FGF and EGF. Cells were expanded in suspension as neurospheres or in adhesion over matrigel during 6-7 passages before RNA extraction for transcriptomic analysis.
Umbilical CB samples were obtained from the Banc de Sang i Teixits, Barcelona. CD34+ cell purification was obtained as previously described (Giorgetti et al., 2009. Cell Stem Cell 5, 353-357). Briefly, mononuclear cells (MNC) were isolated from CB using Lympholyte-H (Cederlane) density gradient centrifugation. CD34+ cells were positively selected using Mini-Macs immunomagnetic separation system (Miltenyi Biotec). Purification efficiency was verified by flow cytometric analysis staining with CD34-phycoerythrin (PE; Miltenyi Biotec) antibody.
Immunofluorescence detection of proteins was performed in cells fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% TritonX-100. Primary and secondary antibodies used are shown in Table 1. Nuclei were stained with a 1 μg/ml solution of 4′,6-diamino-2-phenilindole (DAPI; Life technologies). Fluorescent microscopy was performed in a TiS microscope (Nikon) or in a Leica TCS-SP5. Images were assembled using Adobe® Photoshop® CS5.
Live cells were incubated with primary antibodies (Table 1) for 30 min at 4° C. Fluorescence was estimated with a Macs Quant flow cytometer (Miltenyi) and results were analyzed with MacsQuantify 2.10 software and FloJ v10 software. IgG controls were always runned in parallel with samples. Gating strategy is shown in
RNA was extracted with RNeasy® Mini kit (Quiagen) following the instructions of the manufacturer. Samples were sent to the genomics unit of the Andalusian Molecular Biology and Regenerative Medicine Centre (CABIMER) for the quantification of RNA samples and execution of the expression arrays. RNA quality was analyzed by the Bioanalyzer 2100 (Agilent). Once RNA quality and quantity were confirmed, samples were labelled with biotin and hybridized with independent Human Clarion-S Arrays (Affymetrix). Samples were processed with Affymetrix GeneChip Scanner 7G, the fluidic station 450 of Affymetrix, and the obtained data were analysed with Affymetrix ° GeneChip® Command Console® 2.0 software and R. The microarray expression dataset is publicly available at the GEO repository (https://www.ncbi.nlm.nih.gov/geo/). Further analyses were performed using the Transcriptome Analysis Console (TAC, Affymetrix) v4.0 software and R version 3.5.0. Functional enrichment analysis was performed using the bioinformatics tool EnrichR (http://amp.pharm.mssm.edu/Enrichr/) (Chen et al., 2013 J. Eng. Res. 14; Kuleshov et al., 2016. Nucleic Acids Res. 44). Neuroanatomical references were obtained from the Allen Atlas of the developing human brain.
Cells seeded in matrigel-coated flasks to a cell density of 1,200,000 cells/flask were sent to the Biobank of the Andalusian Sanitary System, were G-banding Karyotyping was performed. Cells were treated with colcemid and potassium chloride and metaphases were treated with trypsin and stained with Giemsa to obtain the G-banding pattern. 15 metaphases were analyzed per cell line. The study was performed according to the International System for Human Cytogenomic Nomenclature (2016).
Data are presented as mean±s.e.m. Significance was determined using one-way analysis of variance (ANOVA) with a Bonferroni post-test or the Student's t-test. All statistical analyses were performed using GraphPad Prism 5.0 software and/or GraphPad Prism 8.01 software. Bioinformatic analyses were performed using Affymetrix and R software with t, ANOVA and repeated measures ANOVA tests and selected thresholds as indicated in the text and figures.
0.1 micrograms of RNA were used for cDNA synthesis using Oligo-dT, RNase OUT™ and SuperScript II Retrotranscriptase (Invitrogen). PCR products were obtained using 5 nanograms of cDNA and Mytaq Red™ DNA Polymerase (Bioline) following instructions of manufacturer. Oligonucleotides used for amplification are described in Table 5.
Transplantation into Nude Mice
Ten nude mice received a single injection of 300,000 CD133+ cells in the striatum. 3 animals were sacrificed at 3 weeks to assess survival and 7 animals at 6 months. Transplantation experiments and analysis were performed as previously described for fetal NSC (Mazzini et al., 2015. J. Transl. Med. 13, 17; Rosati et al., 2018. Cell Death Dis. 9, 937). 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.
26-39 weeks-old premature infants diagnosed with IVH Grade IV according to Papille grading (Papile et al., 1978. J. Pediatr. 92, 529-534) and PHH were treated by endoscopy to seal the injured germinal matrix and to remove hemorrhagic CSF from ventricular cavities, as a measure to reduce the intracerebral pressure and decrease the burden of blood degradation products that may act against the subependymal periventricular area. Neuroendoscopic techniques seem to decrease the need for subsequent shunt procedures and have fewer complications such as infection and development of multi-loculated hydrocephalus. Previous studies suggested that early removal of intraventricular blood degradation products and residual hematoma via neuroendoscopic ventricular irrigation is feasible and safe. Neuroendoscopic lavage was performed following the technique reported by Schulz et al., 2014 disability (Schulz, 2014. J. Neurosurg. Pediatr. 13, 626-635) with some modifications.
The ventricle with the larger amount of hematoma was punctured with the surgical endoscope (Aesculap MInop™) under intraoperative ultrasound guidance. When ventricular cavities were approached, under direct vision continuous irrigation was established using warm lactate-free Ringer solution, by passive inflow via an infusion system through the irrigation channel of the endoscope. Simultaneously, a passive outflow was ensured through the second channel (1.4 mm wide) to balance the intracranial volume and avoid any significant changes in intracranial pressure. This outflow was collected for subsequent recovery of stem cells through a three-way connection attached to 50 ml syringes with luer-lock connection in order to assure sterility and minimal handling of hemorrhagic CSF. Irrigation was stopped once the fluid within the ventricular system was clear or hemodynamic instability appeared during surgery. Typically, 1000-2000 ml of Ringer solution were used and collected in the 50 ml sterile syringes that were immediately closed with a cap to maintain a sterile liquid. Bleeding area was sealed with gelatin beads with thrombin (Floseal, Baxter Healthcare Corporation) (
CSF was centrifuged and the obtained cell suspension was seeded in PLO-laminin or matrigel coated plates (see Experimental procedures for details of the protocol). 24-48 h after seeding the plate was filled of erythrocytes and blood cells in suspension but some groups of cells in the NSC-size and tissue spheres slightly adhered to the plate were observed (
We tried to isolate these NSCs-like cells from samples of non-hemorrhagic cebrospinal fluid and rebleedings of PHH patients (see
The neural progenitor-like cells obtained from hemorrhagic CSF (CSF-NSCs) were analysed by flow cytometry at early (passage 3) and late passage (passage 7). Samples were positive for CD133 and CD24, and negative for CD45, a similar pattern to that published for foetal NSCs, however, CSF-NSCs showed a higher percentage of CD34 positive cells (
CSF-NSCs were analyzed as well by inmunofluorescence for the expression of NSCs (Sox1, Sox2, and Nestin) and fibroblasts (CD13, Collagen I, vimentin and Fibronectin) markers. Cells were positive for all the markers analysed with the exception of fibroblast markers CD13, Collagen I and Fibronectin (
There were no significant differences in marker expression between early and late passage CSF-NSCs (
Eight consecutive cases with a clinical and radiological diagnosis of grade IV IVH (Table 1) underwent a ventricular neuroendoscopy to seal the bleeding GM and remove the hemorrhagic CSF from the ventricular cavities (
After centrifugation, the cell pellet was seeded on poly-L-ornithine/laminin (POL) or matrigel coated plates and cultured in an N2/B27 serum free medium with mitogens. 24-48 h after seeding, small aggregates were observed amidst abundant erythrocytes and blood cells in suspension (
We next analyzed whether the cell population obtained from hemorrhagic CSF had a similar expression pattern of cluster of differentiation (CD) surface antigens than that described for fetal NSC (Tamaki et al., 2002 J. Neurosci. Res. 69, 976-986; Uchida et al., 2000. Proc Natl Acad Sci USA. December 19; 97(26):14720-5). Like fetal NSC, most cells in CSF samples were positive for CD133 and all were negative for CD45, displaying a variable expression of CD24 (
We next confirmed, by immunofluorescence, the expression of radial glia stem cell markers such as SOX2, nestin and brain lipid binding protein (BLBP, FABP7) (
We did not obtain NSC-like cells from non-hemorrhagic CSF samples, although in some cases, changing to a serum-based media allowed us to grow fibroblast-like, adherent cells from samples with large volumes (>20 mL) (
Taken together these experiments confirm that we can isolate NSC from the hemorrhagic CSF of preterm neonates.
To study whether CSF-NSCs were the result of a in vitro-forced differentiation of MSCs or HSCs or on the other hand were NSCs derived from the germinal matrix of PHH patients, we compared CSF-NSCs by transcriptome analyses with bone-marrow derived mesenchymal stem cells (BM-MSCs), cord blood CD34+ cells, iPS derived NSCs and fetal NSCs.
PCA mapping and hierarchical clustering of global gene-expression profiles showed that CSF-NSCs are clustered together with fetal NSCs being farther away from IPS-derived NSCs and stem cells from other sources (
To study in more detail the dynamics of CSF-NSCs in culture, we performed comparative transcriptome analyses of 3 samples of CSF-NSCs at early and late passage finding no significant changes (FDR) in RNA expression between short and long passage in CSF-NSCs nor fetal NSCs (
We next performed a transcriptomic analysis to study the differences and similarities between NSC isolated from hemorrhagic CSF, fetal forebrain NSC and NSC derived from iPSC. Given that CSF samples contained mostly blood cells we also included in the analysis hematopoietic stem cells (CD34+ CB-HSC). Principal component analysis (PCA) mapping and hierarchical clustering of global gene-expression profiles showed that CSF-derived NSC clustered together with fetal NSC, being farther away from iPS-derived NSC (
Consistent with our initial characterization, expression of radial glia and neural progenitor markers, such as SOX2, FABP7, FOXG1, DCX or SOX1 was similar in fetal and CSF-derived NSC (
3. Purification of CD133+ Cells from Hemorrhagic Cebrospinal Fluid-Derived NSCs
To assure NSC purity, CSF-NSCs were screened by magnetic activated cell sorting (MACS) with CD133 beads isolating a population 91.17+/−4.54% CD133+ that still maintain normal morphology (
We examined transcriptomic changes related to propagation and CD133 purification (
We validated the expression of these six candidate genes at the protein level using flow cytometry or immunofluorescence before and after MACS purification.
PODXL is an interesting glycoprotein involved in apical polarity, that belongs to the CD34 family of sialomucins, whose absence has been reported to cause ventricular enlargement in mice (Nowakowski et al., 2010. Mol. Cell. Neurosci. 43, 90-97). PODXL was expressed by nearly all cells in all samples and expression was maintained after CD133 sorting. The interleukin 1 receptor accessory protein (IL1RAP) is differentially expressed in the human VZ (Fietz et al., 2012. Proc. Natl. Acad. Sci. U.S.A. 109, 11836-11841). We confirmed IL1RAP expression in GM-NSC at the protein level by flow cytometry before and after MACS purification (
We used immunofluorescence to study the expression of PLPP4, a poorly characterized phospholipid phosphatase expressed in the brain (Human protein atlas). Despite its predicted membrane localization, GM-NSC showed a PLPP4 nucleolar localization pattern consistent with that described in human cell lines (Human Protein Atlas, www.proteinatlas.org) and this pattern was maintained in sorted CD133+ cells (
Finally, to evaluate the safety of GM-NSC, nude mice were transplanted with CD133+ purified cells in the striatum. GM-NSC remained in the tissue at and around the site of the injection 3 weeks after transplantation, showing no signs of tumor development or uncontrolled proliferation (
| Number | Date | Country | Kind |
|---|---|---|---|
| 18382367.3 | May 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/063888 | 5/28/2019 | WO | 00 |
