Dedicated to the memory of Dr. Rainer Saffrich.
The authors are especially grateful to Jos&#233; Manuel Garc&#237;a-Verdugo and Vicente Herranz-P&#233;rez for performing EM experiments and analysis at the Laboratory of Comparative Neurobiology, Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, CIBERNED, Valencia, Spain, which was supported by research funding from the Prometeo Grant for Excellence Research Groups PROMETEO/2019/075. The rest of this work was supported by ACA CELL Biotech GmbH Heidelberg, Germany. 
&#160;

Ethic approvals were obtained when performing the experiments.
1. Isolation of human peripheral blood mononuclear cells (PBMNCs)
<ol>
	<li>Ensure that all donors signed informed consent before blood withdrawing in compliance with institutional guidelines.</li>
	<li>Take 30 mL of blood from healthy donors by trained medical personnel according to the standard protocol.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Count the number of cells by standard methods using a counting chamber.</li>
</ol>
2. Activation of human GPI-anchored glycoprotein by antibody crosslinking on the surface of PBMNCs
<ol>
	<li>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 &#181;g/mL) for 30 min in PBS with 1% bovine serum albumin (BSA) at 37 &#176;C.</li>
	<li>Replace incubation medium with Iscove&#39;s modified Dulbecco&#39;s medium supplemented with 10% fetal bovine serum (FBS).</li>
	<li>Grow cells in 15 mL polystyrene tubes, put the tubes in an incubator at 37 &#176;C and 5% CO2 for 8-10&#160;days (without shaking). On D5, add an additional 1-2 mL of Iscove&#39;s medium supplemented with 10% FBS to each 15 mL tube.</li>
</ol>
3. Sorting of newly generated dedifferentiated cells
<ol>
	<li>Count cells with an automated cell counter (18 &#181;L cell suspension + 2 &#181;L fluorescence dye) or in a counting chamber.</li>
	<li>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.</li>
	<li>Re-suspend the cell pellet in 90 &#181;L of pre-cooled PBS pH 7.2, 0.5% BSA and 2 mM EDTA.</li>
	<li>Add CD45 positive nano-sized magnetic beads (80 &#181;L) to the cell suspension and incubate on ice for 15 min.</li>
	<li>Wash the cells by adding 2 mL of PBS buffer and centrifuge at 300 x g for 10 min.</li>
	<li>Re-suspend the cells in 500 &#181;L of PBS buffer.</li>
	<li>Wash the column with 500 &#181;L of pre-cooled PBS buffer and place it in the magnetic field.</li>
	<li>Place the cell suspension on the column and wash it with 500 &#181;L of PBS buffer (two times) and the centrifuge flow containing CD45 negative cells. Collect them in Iscove&#39;s medium supplemented with 1% BSA.</li>
	<li>Count the cells in the counting chamber.</li>
</ol>
4. Preparing cell culture dishes for neuronal differentiation of newly generated stem cells
<ol>
	<li>Coat the culture vessels with poly-L-ornithine and laminin for growing neuronal cells.</li>
	<li>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 &#176;C incubator for 1 h. Then wash with ddH2O.</li>
	<li>Slowly thaw laminin (0.5-2.0 mg/mL) and add to the top of coverslips. Incubate it at 37 &#176;C for 2 h.</li>
	<li>Prepare neural induction medium N2 consisting of 49 mL of D-MEM/F12, 500 &#181;L of N2 supplement, 400 &#181;L of non-essential amino acids (NEAA), basic FGF solution at 20 ng/mL final concentration (prepared from 100 &#181;g/mL stock solution), and heparin at 2 ng/mL final concentration.</li>
	<li>Remove excess laminin by pipetting and add neuronal medium N2 to culture dishes.</li>
</ol>
5. Culturing of neuronal dedifferentiated blood cells
<ol>
	<li>Culture BD-derived CD45 negative cells on laminin/ornithine-coated glass coverslips for 2 days in an incubator at 37 &#176;C and 5% CO2 in N2 medium to initiate a neuronal differentiation of newly BD-generated cells.</li>
	<li>Culture cells further in neuronal differentiation medium consisting of 48 mL of Neurobasal medium, 500 &#181;L of L-glutamine, 1 mL of B27 Supplement, 500 &#181;L of NEAA, 50 &#181;L of recombinant human glial-derived neurotrophic factor (GDNF) at 5 &#181;g/250 &#181;L in PBS/0.1% BSA, and 50 &#181;L of recombinant human brain derived neurotrophic factor (BDNF) at 5 &#181;g/200 &#181;L in PBS/0.1% BSA and 50 &#181;L of ascorbic acid solution 2.9 g/50 mL in PBS. Place plates in an incubator at 37 &#176;C and 5% CO2.</li>
</ol>
6. Immunofluorescence microscopy analysis of blood-derived neural cells
<ol>
	<li>Culture the cells as described above for 16 days and remove the media.
	<ol>
		<li>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 MgCl2, 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.</li>
		<li>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.</li>
		<li>Block the cells at room temperature on a rocker plate for 1 hour.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
7. Transmission electron microscopy analysis of newly generated cells
<ol>
	<li>Seed the cells for TEM in 8-well chamber slides.</li>
	<li>Fix the cells in 3.5% glutaraldehyde for 1 h at 37 &#176;C, post-fix in 2% OsO4 for an additional hour at room temperature and stain in 2% uranyl acetate in the dark at 4 &#176;C for 2 h 30 min.</li>
	<li>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 &#176;C oven for 72 h for resin hardening.</li>
	<li>Detach the embedded cell cultures from chamber slide and glue to araldite blocks.</li>
	<li>Cut serial semi-thin sections (1.5 &#181;m) with a machine, mount onto glass-slides and lightly stain with 1% toluidine blue.</li>
	<li>Glue selected semi-thin sections to araldite blocks and detach them from the glass slide by repeated freezing (in liquid nitrogen) and thawing.</li>
	<li>Prepare ultrathin sections (0.06-0.08 &#181;m) with a machine and further contrast with lead citrate.</li>
	<li>Obtain micrographs by using electron scanning microscope with digital camera.</li>
</ol>

<ol>
	<li>Peng, J., Zeng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Induced+Pluripotent+Stem+Cells+in+Regenerative+Medicine:+Neurodegenerative+Diseases.">The Role of Induced Pluripotent Stem Cells in Regenerative Medicine: Neurodegenerative Diseases.</a> Stem Cell Research and Therapy. 2 (4), 32 (2011).</li><li>Sorells, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hippocampal+neurogenesis+drops+sharply+in+children+to+undetectable+levels+in+adults.">Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults.</a> Nature. 555 (7696), 377-381 (2018).</li><li>Thomson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts.">Embryonic Stem Cell Lines Derived from Human Blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Pluripotent+Stem+Cells+From+Adult+Human+Fibroblast+by+Defined+Factors.">Induction of Pluripotent Stem Cells From Adult Human Fibroblast by Defined Factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Liu, G. -. H., Yi, F., Suzuki, K., Qu, J., Izpisua Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+neural+stem+cells:+a+new+tool+for+studying+neural+development+and+neurological+disorders.">Induced neural stem cells: a new tool for studying neural development and neurological disorders.</a> Cell Research. 22 (7), 1087-1091 (2012).</li><li>Becker-Koji&#263;, Z. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+by+ACA+Induces+Pluripotency+in+Human+Blood+Progenitor+Cells.">Activation by ACA Induces Pluripotency in Human Blood Progenitor Cells.</a> Cell Technologies in Biology and Medicine. 2, 85-101 (2013).</li><li>Marchenko, S., Flanagan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemistry:+Human+Neural+Stem+Cells.">Immunocytochemistry: Human Neural Stem Cells.</a> Journal of Visualized Experiments. , e267 (2007).</li><li>Becker-Koji&#263;, Z. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+glycoprotein+ACA+is+upstream+regulator+of+human+heamtopoiesis.">A novel glycoprotein ACA is upstream regulator of human heamtopoiesis.</a> Cell Technologies in Biology and Medicine. 2, 69-84 (2013).</li><li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurochemical+Regulation+of+the+Expression+and+Function+of+Glial+Fibrillary+Acidic+Protein+in+Astrocytes.">Neurochemical Regulation of the Expression and Function of Glial Fibrillary Acidic Protein in Astrocytes.</a> Glial. 68 (5), 878-897 (2020).</li><li>Melkov&#225;, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Functions+of+Microtubule+Associated+Proteins+Tau+and+MAP2c:+Similarities+and+Differences.">Structure and Functions of Microtubule Associated Proteins Tau and MAP2c: Similarities and Differences.</a> Biomolecules. 9 (3), 105 (2019).</li><li>Menezes, J. R., Luskin, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+neuron-specific+tubulin+defines+a+novel+population+in+the+proliferative+layers+of+the+developing+telencephalon.">Expression of neuron-specific tubulin defines a novel population in the proliferative layers of the developing telencephalon.</a> Journal of Neuroscience. 14 (9), 5399-5416 (1994).</li><li>Bernal, A., Arranz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nestin-expressing+progenitor+cells:+function,+identity+and+therapeutic+implications.">Nestin-expressing progenitor cells: function, identity and therapeutic implications.</a> Cellular and Molecular Life Sciences. 75 (12), 2177-2195 (2018).</li></ol>

The corresponding author declares that she is a patent holder related to Novel Human GPI-linked Protein as well as she co-founded and works for ACA CELL Biotech. The other authors declare that they do not have any conflict of interest.

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&#39;s disease, Alzheimer&#39;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.

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 PLC&#947;/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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Albumin Fraction V</td>
			<td>Roth</td>
			<td>T8444.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFAP Cy3 conjugate</td>
			<td>Merck Millipore</td>
			<td>MAB3402C3</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2 Alexa Fluor 555</td>
			<td>Merck Millipore</td>
			<td>MAB3418A5</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nestin Alexa Fluor 488</td>
			<td>Merck Millipore</td>
			<td>MAB5326A4</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Tuj1 Alexa Fluor 488</td>
			<td>BD Pharmingen</td>
			<td>560381</td>
			<td></td>
		</tr>
		<tr>
			<td>AO/PI Cell Viability kit</td>
			<td>Biozym</td>
			<td>872045</td>
			<td>Biozym discontinued. The product produced by Logos Biosystems.</td>
		</tr>
		<tr>
			<td>Ascorbic acid 2-phosphate sequimagnes</td>
			<td>Sigma Aldrich</td>
			<td>A8960-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Serum free 50x</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>11530536</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic FGF solution</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>10647225</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocoll</td>
			<td>Merck Millipore</td>
			<td>L6115-BC</td>
			<td>density gradient media</td>
		</tr>
		<tr>
			<td>BSA Frac V 7.5%</td>
			<td>Gibco</td>
			<td>15260037</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-045-801</td>
			<td>nano-sized magnetic beads</td>
		</tr>
		<tr>
			<td>Cell counting slides Luna</td>
			<td>Biozym</td>
			<td>872010</td>
			<td>Biozym discontinued. The product produced by Logos Biosystems.</td>
		</tr>
		<tr>
			<td>Chamber Slides Lab-Tek</td>
			<td>Fisher Scientific</td>
			<td>10234121</td>
			<td></td>
		</tr>
		<tr>
			<td>D-MEM/F12</td>
			<td>Merck Millipore</td>
			<td>FG4815-BC</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan</td>
			<td>Sigma Aldrich</td>
			<td>44610</td>
			<td>epoxy resin</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Merck Millipore</td>
			<td>S0115/1030B</td>
			<td>Discontinued. Available under: TMS-013-B</td>
		</tr>
		<tr>
			<td>GDNF recombinant human</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>10679963</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax 100x</td>
			<td>Gibco</td>
			<td>35050038</td>
			<td>L-glutamine</td>
		</tr>
		<tr>
			<td>Glutaraldehyde grade</td>
			<td>Sigma-Aldrich</td>
			<td>G5882-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium cell</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-50KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BDNF</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>11588836</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove (IMDM)</td>
			<td>Biochrom</td>
			<td>FG0465</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin mouse</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>10267092</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead citrate</td>
			<td>Sigma-Aldrich</td>
			<td>15326-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Luna FL Automated Cell Counter</td>
			<td>Biozym</td>
			<td>872040</td>
			<td>Biozym discontinued. The product produced by Logos Biosystems.</td>
		</tr>
		<tr>
			<td>MACS Buffer</td>
			<td>Miltenyi</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA 100x</td>
			<td>Gibco</td>
			<td>11140035</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Trenns&#228;ulen</td>
			<td>Miltenyi</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Morada digital camera</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiplatte Nunclon 4 wells</td>
			<td>Fisher Scientific</td>
			<td>10507591</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement 100x</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>11520536</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>10888022</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS sterile</td>
			<td>Roth</td>
			<td>9143.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma-Aldrich</td>
			<td>P4957-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Glue-3 Loctite</td>
			<td>Henkel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEM FEI Technai G2 Spirit</td>
			<td>FEI Europe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracut UC-6</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate C</td>
			<td>EMS</td>
			<td>22400</td>
			<td></td>
		</tr>
	</tbody>
</table>

gm-free generation of blood-derived neuronal cells

Many human&#160;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.

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 PLC&#947;/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-PSCs6,8.
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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61634/61634fig01.jpg" /> 
Figure 1: Generation of dedifferentiated (pluripotent) stem cells.&#160;(A) Ficoll-isolated mononuclear cells were grown in Iscove&#39;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 &#181;m. (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.&#160;<a href="https://www.jove.com/files/ftp_upload/61634/61634fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61634/61634fig02.jpg" /> 
Figure 2: Neuro re-differentiation of BD-PSCs.&#160;(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 &#181;m. (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 &#181;m; (b-d), 500 nm.&#160;<a href="https://www.jove.com/files/ftp_upload/61634/61634fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61634/61634fig03.jpg" /> 
Figure 3: Immunophenotyping of newly generated neuronal cells.&#160;BD-dedifferentiated cells&#160;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 &#181;m. Control is presented in Supplementary Figure 1.&#160;<a href="https://www.jove.com/files/ftp_upload/61634/61634fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: BD-dedifferentiated cells control. BD-derived undifferentiatedcells were cultured in Iscove&#39;s modified Dulbecco&#39;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 &#181;m.&#160;<a href="https://www.jove.com/files/ftp_upload/61634/Supplementary Figure 1.psd" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about GM-Free Generation of Blood-Derived Neuronal Cells at JoVE.com

GM-Free Generation of Blood-Derived Neuronal Cells

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.

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

gm-free-generation-of-blood-derived-neuronal-cells

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

2.8K Views.  ACA CELL Biotech GmbH. 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. 

Video: GM-Free Generation of Blood-Derived Neuronal Cells

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...