GM-Free Generation of Blood-Derived Neuronal Cells

Summary

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Abstract

Many human neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

Introduction

Development of basic and pre-clinical stem cell research methods encourages the clinical application of stem cell-based therapies for neurological diseases. Such potential therapy critically depends on the method for generation of human neural cells leading to functional recovery¹.

Neural stem cells (NSCs) self-renew and differentiate into new neurons throughout life in a process called adult neurogenesis. Only very restricted brain areas harbor NSCs competent to generate newborn neurons in adulthood. Such NSCs can give rise to mature neurons, which are involved in learning and memory, thus replacing lost or damaged neurons. Unfortunately, these NSCs are present in restricted amounts and this limited neurogenesis decreases rapidly during juvenile development². Therefore, other sources of neural cells must be considered in a cell therapy objective.

Degenerative neurological diseases are difficult to cure using standard pharmacological approaches. New therapeutic strategies for embracing many immedicable neurological disorders are based on cell replacement therapies of diseased and injured tissue. NSC transplantation could replace damaged cells and provide beneficial effects. Other sources for neural cell replacement include human embryonic stem cells (ESC), which are derived from the inner cell mass of mammalian blastocysts³, as well as iPSCs⁴, which have extensive self-renewal capacity like ESCs and are capable to differentiate into various cell lineages. NSCs can also be generated by direct reprogramming from human fibroblasts avoiding pluripotent state⁵.

Cell replacement therapy is still a challenging issue. Though ESC, fetal, or iPS can be a source for generation of neuronal cells for treating many incurable neurological diseases, autologous adult SCs cell replacement of damaged tissues is a better alternative that circumvents immunological, ethical and safety concerns.

Activation of human GPI-linked protein by antibody-crosslinking via phosphorylation of PLCγ/PI3K/Akt/mTor/PTEN initiates a dedifferentiation of blood progenitor cells and generation of blood-derived pluripotent stem cells (BD-PSCs)⁶. These cells differentiate in vitro toward neuronal cells as confirmed by means of brightfield, immunofluorescence and transmission electron microscopy (TEM) analysis.

In this work we describe the GM-free generation of BD-PSCs and their successful re-differentiation into cells with neuronal phenotype.

Protocol

Ethic approvals were obtained when performing the experiments.

1. Isolation of human peripheral blood mononuclear cells (PBMNCs)

1. Ensure that all donors signed informed consent before blood withdrawing in compliance with institutional guidelines.
2. Take 30 mL of blood from healthy donors by trained medical personnel according to the standard protocol.
3. Isolate PBMNCs by density gradient media. Use 10 mL of media with 25 mL of 1:1 blood diluted with phosphate buffer saline (PBS), and centrifuge at 300 x g for 30 min.
4. Isolate the interphase layer between the plasma and the density gradient media by pipetting. Wash the isolated cells with 5 mL of sterile PBS and centrifuge at 300 x g for 10 min. Repeat twice.
5. Count the number of cells by standard methods using a counting chamber.

2. Activation of human GPI-anchored glycoprotein by antibody crosslinking on the surface of PBMNCs

1. Place the 6 x 10⁶ mononuclear cells (MNCs) in 15 mL tubes and perform antibody crosslinking by incubating the cells with human GPI-linked membrane protein-specific antibody (30 µg/mL) for 30 min in PBS with 1% bovine serum albumin (BSA) at 37 °C.
2. Replace incubation medium with Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum (FBS).
3. Grow cells in 15 mL polystyrene tubes, put the tubes in an incubator at 37 °C and 5% CO₂ for 8-10 days (without shaking). On D5, add an additional 1-2 mL of Iscove's medium supplemented with 10% FBS to each 15 mL tube.

3. Sorting of newly generated dedifferentiated cells

1. Count cells with an automated cell counter (18 µL cell suspension + 2 µL fluorescence dye) or in a counting chamber.
2. Centrifuge cultured cell suspension (5-7 x 10⁶) at 300 x g for 10 min and aspirate the resulting supernatant with a sterile Pasteur pipette.
3. Re-suspend the cell pellet in 90 µL of pre-cooled PBS pH 7.2, 0.5% BSA and 2 mM EDTA.
4. Add CD45 positive nano-sized magnetic beads (80 µL) to the cell suspension and incubate on ice for 15 min.
5. Wash the cells by adding 2 mL of PBS buffer and centrifuge at 300 x g for 10 min.
6. Re-suspend the cells in 500 µL of PBS buffer.
7. Wash the column with 500 µL of pre-cooled PBS buffer and place it in the magnetic field.
8. Place the cell suspension on the column and wash it with 500 µL of PBS buffer (two times) and the centrifuge flow containing CD45 negative cells. Collect them in Iscove's medium supplemented with 1% BSA.
9. Count the cells in the counting chamber.

4. Preparing cell culture dishes for neuronal differentiation of newly generated stem cells

1. Coat the culture vessels with poly-L-ornithine and laminin for growing neuronal cells.
2. Place the glass coverslips in 4-well plates and coat it with 1:5 diluted poly-L-ornithine (0.1 mg/mL in ddH₂O) in ddH₂O. Place the coverslips into a 37 °C incubator for 1 h. Then wash with ddH₂O.
3. Slowly thaw laminin (0.5-2.0 mg/mL) and add to the top of coverslips. Incubate it at 37 °C for 2 h.
4. Prepare neural induction medium N2 consisting of 49 mL of D-MEM/F12, 500 µL of N2 supplement, 400 µL of non-essential amino acids (NEAA), basic FGF solution at 20 ng/mL final concentration (prepared from 100 µg/mL stock solution), and heparin at 2 ng/mL final concentration.
5. Remove excess laminin by pipetting and add neuronal medium N2 to culture dishes.

5. Culturing of neuronal dedifferentiated blood cells

1. Culture BD-derived CD45 negative cells on laminin/ornithine-coated glass coverslips for 2 days in an incubator at 37 °C and 5% CO₂ in N2 medium to initiate a neuronal differentiation of newly BD-generated cells.
2. Culture cells further in neuronal differentiation medium consisting of 48 mL of Neurobasal medium, 500 µL of L-glutamine, 1 mL of B27 Supplement, 500 µL of NEAA, 50 µL of recombinant human glial-derived neurotrophic factor (GDNF) at 5 µg/250 µL in PBS/0.1% BSA, and 50 µL of recombinant human brain derived neurotrophic factor (BDNF) at 5 µg/200 µL in PBS/0.1% BSA and 50 µL of ascorbic acid solution 2.9 g/50 mL in PBS. Place plates in an incubator at 37 °C and 5% CO₂.

6. Immunofluorescence microscopy analysis of blood-derived neural cells

1. Culture the cells as described above for 16 days and remove the media.
   1. Incubate with pre-warmed fixative consisting of 75 mL of sterile water, 4 g of paraformaldehyde. Add 10 N NaOH as needed and stir until the solution clears. Then add 10 mL of 10x PBS, 0.5 mL of MgCl_2, 2 mL of 0.5 M EGTA, and 4 g of sucrose. Titrate to pH 7.4 with 6 N HCl, and bring to 100 mL of sterile water for 15 min, according to Marchenko et al.⁷.
   2. Discard the fixative and wash the cells 3 times for 5 min each time. Immediately add a freshly made 0.3% Triton X-solution and permeabilize the cells for 5 min. Wash 3 times with PBS and add a blocking solution made by PBS and 5% BSA.
   3. Block the cells at room temperature on a rocker plate for 1 hour.
   4. Prepare appropriate dilution of antibodies in 1% BSA/PBS and incubate the cells with antibody dilutions on rocker plate for 1.5 h at room temperature. Wash the cells 3 times with PBS for 5 min each, incubate the cells with DAPI and mount the coverslips with mounting media for visualization on a microscope.
      NOTE: Directly labeled antibodies used in this experiment are listed in Table of Materials.

7. Transmission electron microscopy analysis of newly generated cells

1. Seed the cells for TEM in 8-well chamber slides.
2. Fix the cells in 3.5% glutaraldehyde for 1 h at 37 °C, post-fix in 2% OsO₄ for an additional hour at room temperature and stain in 2% uranyl acetate in the dark at 4 °C for 2 h 30 min.
3. Finally, rinse the cells in distilled water, dehydrate it in ethanol and embed in epoxy resin overnight. The following day transfer the samples to a 70 °C oven for 72 h for resin hardening.
4. Detach the embedded cell cultures from chamber slide and glue to araldite blocks.
5. Cut serial semi-thin sections (1.5 µm) with a machine, mount onto glass-slides and lightly stain with 1% toluidine blue.
6. Glue selected semi-thin sections to araldite blocks and detach them from the glass slide by repeated freezing (in liquid nitrogen) and thawing.
7. Prepare ultrathin sections (0.06-0.08 µm) with a machine and further contrast with lead citrate.
8. Obtain micrographs by using electron scanning microscope with digital camera.

Representative Results

The results provide evidence that this novel GM-free method is capable of reverting blood progenitor cells to their most primitive state without directly acting on the human genome.

We have previously shown that GPI-linked protein specific antibody crosslinking initiates via PLCγ/IP3K/Akt/mTOR/PTEN upregulation of highly conserved developmentally relevant genes such as WNT, NOTCH and C-Kit, thus initiating a process of dedifferentiation that leads to the first step to generation of HSCs and a second and final to a generation of BD-PSCs^6,8.

Activated MNC cultures were subjected to immunomagnetic sorting using CD45 microbeads. Mature blood cells that cannot be reprogrammed with this method (e.g., CD45 positive cells) were retained on the column, whereas the negative fraction containing reprogrammed cells (CD45 negative cells) was used for generation of various neuronal lineage cells.

We first studied the morphological aspects of peripheral BD-dedifferentiated cells by means of light and TEM. As shown in Figure 1, specific GPI-anchored glycoprotein antibody crosslinking of human MNCs generates a steady growing new population of cells (Figure 1A). We analyzed these cells by means of TEM. BD-dedifferentiated cells are small in size and show the characteristics of immature agranular cells, with gradually less organelles and large nuclei with condensed chromatin, similar to ESCs (Figure 1B). Non-treated cultures showed a trend towards a gradual disappearance.

BD-CD45 negative cells were subjected to neuronal differentiation in two steps. We initiated the differentiation towards neuronal lineages by seeding the CD45 negative cells on poly-L-ornithine/laminin coated culture plates for 2 days in N2 medium following culture in neuronal differentiation medium. Brightfield pictures were acquired at days 4, 8, 10, 14 and 30 respectively upon starting neuronal differentiation of BD-generated stem cells.

As early as 4 days after starting the targeted differentiation of newly generated cells, the first neuronal-like cells with long branching structures could be detected. We observe the morphological changes from D2 to D30 with a more complex structure including ramification, implying an active process toward differentiation to neuronal lineages throughout the culture time period (Figure 2). To confirm the neuronal features of re-differentiated cells after culturing them in neuronal medium for 16 days, cells were fixed according to a previous protocol⁷, and immunocytochemistry (ICC) was performed using antibody detection to nestin, glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2) and neuron-specific class III beta-tubulin (Tuj1).

GFAP is the protein that constitutes a portion of cytoskeleton in astrocytes representing the principal intermediate filament of mature astrocytes. As shown in Figure 3, the antibody to GFAP recognizes these structures in the newly generated neuronal cells, confirming that BD-PSCs are capable of re-differentiation towards human astrocytes⁹.

MAP2 is a cytoskeleton protein that binds to tubuli and stabilizes microtubules. It is expressed within axons, dendrites and cell bodies and this expression is tissue- and developmentally-specific. The immunofluorescence microscopy results confirm the expression of this protein in re-differentiated cells¹⁰.

Tuj1 is typical neuronal cell marker. Its function is to stabilize microtubili in neuronal cell body and axons. It is also implicated in axonal transport¹¹. Newly re-differentiated cells clearly confirmed the expression of this protein at D16 upon starting the neuronal differentiation under the condition described here.

Nestin was first characterized in NSCs and represents a neuro epithelial stem cell protein, which belongs to intermediate filament (IF) protein¹² distinguishing neuronal progenitor cells from more differentiated neuronal cells. These IF proteins are expressed mostly in nerve cells where they are involved in the radial growth of the axon, but they are also present in a number of additional tissues. Nestin as a marker of predominantly NSCs is weakly expressed in the cells already on the path to differentiate into specific neuronal lineages as it is the case with BD-re-differentiated cells at D16.

Figure 1: Generation of dedifferentiated (pluripotent) stem cells. (A) Ficoll-isolated mononuclear cells were grown in Iscove's medium supplemented with 10% FBS. Micrographs of activated cultured cells were taken at days 1, 5 and 10, respectively. Non-activated MNCs were studied as a control. Scale bar: 50 µm. (B) TEM analysis of newly generated cells throughout culture time shows that organelles of mature cells (D1) gradually disappear (D8), leading to generation of completely dedifferentiated cells resembling ESCs. Please click here to view a larger version of this figure.

Figure 2: Neuro re-differentiation of BD-PSCs. (A) BD-dedifferentiated cells were placed in ornithine/laminin coated culture dishes and cultured for 30 days as described in protocol. Micrographs are taken at days 4, 8, 10, 14, and 30 respectively, after growing in neuronal differentiation conditions. Most cells in the culture changed their morphology from small spherical shapes to larger, elongated shapes and in some cases branched cells. Scale bar: 100 µm. (B) BD-dedifferentiated cells were grown for 16 days in neuronal medium, fixed in glutaraldehyde and EM analysis performed as described in protocol section. The cell body and processes of these cells showed a higher complexity than those of undifferentiated cell in terms of organelles and cytoskeleton presenting a high density of stacked cisternae of rough endoplasmic reticulum and abundant bundles of actin filaments (a, b). Unlike undifferentiated BD-cells, cells growing in differentiation media frequently established cell-to-cell contacts. Some of these specialized contacts involved cellular body (c) while others involved cellular processes in neurite-like fashion (d). Scale bars: (a) 20 µm; (b-d), 500 nm. Please click here to view a larger version of this figure.

Figure 3: Immunophenotyping of newly generated neuronal cells. BD-dedifferentiated cells were cultured as described in the protocol for 16 days and immunocytochemistry analysis was performed using antibodies to neuronal markers nestin, GFAP, MAP2 and Tuj1. Shown are brightfield micrographs of re-differentiated cells accompanied with immunofluorescence pictures with DAPI as nuclear staining, as well as staining with relevant antibodies. Depicted are the fields showing a particular population that expresses one of the specific neuronal marker characteristic for specific lines. Scale bar: 100 µm. Control is presented in Supplementary Figure 1. Please click here to view a larger version of this figure.

Supplementary Figure 1: BD-dedifferentiated cells control. BD-derived undifferentiatedcells were cultured in Iscove's modified Dulbecco's medium supplemented with 10% FBS for 16 days as described in protocol and stained with antibody to nestin GFAP, Tuj1 and MAP2. DAPI was used for nuclear staining. Scale bar: 100 µm. Please click here to download this File.

Discussion

The non-GM method of reprogramming human cells described in this work is based on membrane to nucleus activation of signaling(s) machinery behind the GPI-linked human membrane glycoprotein that initiates the process of dedifferentiation leading to the ex vivo generation and expansion of self-renewing PSCs obtained from non-manipulated human peripheral blood. These cells when cultured in appropriate media are capable of re-differentiation into cells belonging to different germ layers⁶.

The data presented in this work show that GM-free generated BD-PSCs cells when cultured in a neuronal differentiation media acquired a completely different phenotype, with elongated shapes, higher development of their organelles and established more complex interactions between cells. Moreover, re-differentiation using condition described here, implies neuronal differentiation towards various neuronal lineages.

To obtain the optimal number and the best quality of reprogrammed cells for their use in re-differentiation studies, fresh MNC preparations might be advantageous when compared to frozen MNC preparations. The method of immunomagnetic sorting that separated BD-PSCs from terminally differentiated cells that cannot be reprogrammed by this method could be incomplete requiring that the procedure be repeated, which is very stressful for cells and results in their premature deaths.

The critical step within the protocol relates to the number and quality of MNCs that could be obtained by the method described. Modification of the neural differentiation media as well as culture time can improve the differentiation potential of BD-PSCs, thus leading to generation of specific types of neuronal cells.

A limitation of this method is the non-teratogenic nature of these reprogrammed cells as it is not possible to generate the cell lines with this method. Once the dedifferentiated cells have reached the final stage, that of pluripotency, they become mostly quiescent and a new portion of MNCs must be dedifferentiated again to obtain a higher number of BD-PSCs.

Reprogramming described here relies on antibody crosslinking activation on the surface of blood progenitor cells. This paradigm provides numerous potential advantages with respect to clinical safety when compared to GM methods. The goal of achieving autologous stem cells for generation of neural tissue(s) can be achieved by minimally ex vivo manipulation; therefore strongly suggesting that this cell therapy could be a promising candidate for efficient and safe clinical approach in neurology.

Parkinson's disease, Alzheimer's disease and cerebral ischemia are among the diseases with the highest social and economic burden for the society in Europe and worldwide. The burden of neurodegenerative disorders is expected to increase with the aging population, becoming an important socio-economic problem and creating a desperate need for an answer to the problem. The presented method opens a new avenue for non-invasive therapeutic strategies by utilizing a simple and cost-effective procedure for generating suitable autologous stem cell populations holding a hope for the cure of currently intractable neurological diseases.

Materials

Name	Company	Catalog Number	Comments
Albumin Fraction V	Roth	T8444.4
Anti-GFAP Cy3 conjugate	Merck Millipore	MAB3402C3
Anti-MAP2 Alexa Fluor 555	Merck Millipore	MAB3418A5
Anti-Nestin Alexa Fluor 488	Merck Millipore	MAB5326A4
Anti-Tuj1 Alexa Fluor 488	BD Pharmingen	560381
AO/PI Cell Viability kit	Biozym	872045	Biozym discontinued. The product produced by Logos Biosystems.
Ascorbic acid 2-phosphate sequimagnes	Sigma Aldrich	A8960-5G
B27 Serum free 50x	Fisher Scientific (Gibco)	11530536
Basic FGF solution	Fisher Scientific (Gibco)	10647225
Biocoll	Merck Millipore	L6115-BC	density gradient media
BSA Frac V 7.5%	Gibco	15260037
CD45 MicroBeads	Miltenyi	130-045-801	nano-sized magnetic beads
Cell counting slides Luna	Biozym	872010	Biozym discontinued. The product produced by Logos Biosystems.
Chamber Slides Lab-Tek	Fisher Scientific	10234121
D-MEM/F12	Merck Millipore	FG4815-BC
Durcupan	Sigma Aldrich	44610	epoxy resin
FBS	Merck Millipore	S0115/1030B	Discontinued. Available under: TMS-013-B
GDNF recombinant human	Fisher Scientific (Gibco)	10679963
GlutaMax 100x	Gibco	35050038	L-glutamine
Glutaraldehyde grade	Sigma-Aldrich	G5882-50ML
Heparin sodium cell	Sigma-Aldrich	H3149-50KU
Human BDNF	Fisher Scientific (Gibco)	11588836
Iscove (IMDM)	Biochrom	FG0465
Laminin mouse	Fisher Scientific (Gibco)	10267092
Lead citrate	Sigma-Aldrich	15326-25G
Luna FL Automated Cell Counter	Biozym	872040	Biozym discontinued. The product produced by Logos Biosystems.
MACS Buffer	Miltenyi	130-091-221
MEM NEAA 100x	Gibco	11140035
MiniMACS Trennsäulen	Miltenyi	130-042-201
Morada digital camera	Olympus
Multiplatte Nunclon 4 wells	Fisher Scientific	10507591
N2 Supplement 100x	Fisher Scientific (Gibco)	11520536
Neurobasal Medium	Gibco	10888022
PBS sterile	Roth	9143.2
Poly-L-ornithine	Sigma-Aldrich	P4957-50ML
Super Glue-3 Loctite	Henkel
TEM FEI Technai G2 Spirit	FEI Europe
Ultracut UC-6	Leica
Uranyl acetate C	EMS	22400