Name: Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells (Video) | JoVE Journal
Uploaded: 2015-01-28T15:00:00.000+00:00
Duration: 12 min 6 s
Description: Learn to directly induce human neural stem cells from peripheral blood CD34+ hematopoietic progenitors using Sendai virus transfection and selective medium. This method bypasses iPS generation for patient-specific neural cultures.

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5 minutes

The authors have no conflicts of interest to disclose.

1. Enrichment of Hematopoietic Progenitor Cells from Adult Whole Blood
NOTE: Hematopoietic progenitor cells or CD34+ cells can be purified from peripheral blood mononuclear cells (PBMCs) derived from a variety of sources including cord blood, leukapheresis material and whole blood using density gradient centrifugation based methods. The method listed here uses whole blood as an example.
<ol>
	<li>Isolation of PBMCs from Whole Blood:
	<ol>
		<li>Add 4.5 ml of lymphocyte separation medium to the SepMate tube by pipetting it through the central hole of the insert.</li>
		<li>Dilute the blood sample with an equal volume of sterile DPBS containing 2% human serum (v/v). For example, dilute 5 ml of sample with 5 ml of DPBS + 2% human serum.</li>
		<li>Add the diluted sample by pipetting it down the side of the tube. Take care not to pour the sample directly through the central hole.</li>
		<li>Centrifuge at 1,200 x g for 10 min at RT.</li>
		<li>Carefully remove the supernatant above the PMBC layer.</li>
		<li>Collect the PBMC layer (cloudy) into a new tube.</li>
		<li>Add 15 ml of Dulbecco&#8217;s Phosphate-buffered Saline (DPBS) containing 2% human serum into the tube and centrifuge at 150 x g for 10 min at RT with the brake off.</li>
		<li>Discard the supernatant and resuspend the pellet in 15 ml of DPBS containing 2% human serum. Count the number of cells.</li>
		<li>Centrifuge at 690 x g for 10 min at RT. Remove all the supernatant from the tube.</li>
		<li>Resuspend cells at 1 x 107 cells per 15 ml of Iscove&#8217;s modified Dulbecco&#8217;s medium (IMDM) containing 1% antibiotics (v/v) and 10% human serum (v/v).</li>
		<li>Incubate the cells O/N at 37 &#176;C in 5% CO2 incubator before isolating CD34+ cells.</li>
	</ol>
	</li>
	<li>Positive Selection of CD34+ cells from PBMCs using CD34 MultiSort Kit: 
	NOTE: Work fast and use pre-cooled solutions to make cells cold and prevent capping of antibodies on the cell surface and non-specific cell labeling per the kit instruction.
	<ol>
		<li>Collect all PMBCs in a 50 ml tube and count the number of total cells in the medium.</li>
		<li>Centrifuge the cells at 300 x g for 10 min. Remove all the supernatant completely.</li>
		<li>Resuspend up to 108 cells per 300 &#181;l in bead buffer (DPBS containing 0.5% human serum (v/v) and 2 mM EDTA).</li>
		<li>Add 100 &#181;l of FcR Blocking Reagent and add 100 &#181;l of CD34 MultiSortMicroBeads.</li>
		<li>Mix well and incubate for 30 min in the refrigerator (2 - 8 &#176;C).</li>
		<li>Wash cells by adding 2 ml of bead buffer per 108 cells and centrifuge at 300 x g for 10 min at RT. Remove supernatant completely.</li>
		<li>Resuspend up to 108 cells in 500 &#181;l of buffer.</li>
		<li>Place MACS LS column in the magnetic field of a MACS Separator and prepare column by rinsing with 3 ml of bead buffer.</li>
		<li>Apply cell suspension onto the center of the column. Unlabeled cells will flow through which can be collected if preferred.</li>
		<li>Wash column three times with 3 ml of bead buffer.</li>
		<li>Remove the column from the separator and place it on a 15 ml tube.</li>
		<li>Add 5 ml of bead buffer onto the column and immediately flush out the magnetically labeled cells by pushing the plunger into the column.</li>
		<li>Add 20 &#181;l of MultiSort Release Reagent per 1 ml of cell suspension. Mix well and incubate for 10 min in the refrigerator (2 - 8 &#176;C).</li>
		<li>Add 1 - 2 ml of bead buffer per 107 cells and centrifuge the cells at 300 x g for 10 min at RT. Remove supernatant completely.</li>
		<li>Resuspend cells in 50 &#181;l of bead buffer per 107 cells.</li>
		<li>Add 30 &#181;l of MultiSort Stop Reagent per 107 cells and incubate for 15 min in the refrigerator.</li>
		<li>Add 5 ml of bead buffer and centrifuge the cells at 300 x g for 10 min at RT. Discard the supernatant.</li>
	</ol>
	</li>
</ol>
2. Derivation of Induced Neural Stem Cells from CD34+ Cells
<ol>
	<li>CD34+ cells culture: 
	NOTE: Freshly prepared medium should be used within 7 days when stored in the refrigerator. Significant CD34 cells loss could be observed after 24 hr but the significant proliferation of cells should be noticeable, especially after day 5. If the cell number decreases continuously or there is no proliferation, it implies either bad quality of medium or CD34 cells and the cells should not be used for next step.
	<ol>
		<li>Resuspend and culture CD34+ cells in CD34 maintaining medium (StemSpan SFEM medium containing human thrombopoietin (TPO, 100 ng/ml), fms-like tyrosine kinase 3 (Flt-3) ligand (100 ng/ml) and stem cell factor (SCF, 100 ng/ml), interleukin-6 (IL-6, 20 ng/ml) and interleukin-7 (IL-7, 20 ng/ml)) in a 24-well plate (up to 3 x 105/well in 1 ml of medium per well) at 37 &#176;C in 5% CO2 incubator.</li>
		<li>After 24 hr, collect the floating cells and centrifuge the cells at 110 x g at RT to get rid of any attached cells and cell debris. Discard the supernatant and resuspend the cells in 1 ml of fresh CD34 maintaining medium and seed in a new 24-well plate (up to 1 x 105/well) at 37 &#176;C in 5% CO2 incubator for 5 additional days.</li>
		<li>Replace half of the medium every the other day by carefully aspirating the top half of supernatant while the CD34+ cells are floating on the bottom of the wells (Figure 1A).</li>
	</ol>
	</li>
	<li>Sendai virus transfection:
	<ol>
		<li>On day 5, collect CD34 cells and centrifuge the cells at 170 x g for 10 min at RT. Discard the supernatant.</li>
		<li>Resuspend the cells in fresh medium at 1 x 105/well in a 24-well plate in 1 ml of medium.</li>
		<li>Thaw a cytotune-iPS Sendai reprogramming kit by first immersing the bottoms of the tubes in a 37 &#176;C water bath for 10 sec and then completely thaw at RT. Briefly centrifuge the tubes and put them on ice. Prepare the Sendai virus mixture by mixing the recommended volume of each virus listed on the Certificate of Analysis (COA) for the corresponding lot. MOI 15 is recommended.</li>
		<li>Add Sendai virus mix to each well of CD34 cells. Mix the wells by gently shaking. Incubate the cells at 37 &#176;C in 5% CO2 incubator.</li>
		<li>Observe the transfection efficiency after 24 hr. Notice that cell aggregates and sphere formation are indicators for successful transfection (Figure 1B).</li>
		<li>Change half of the medium by carefully transferring the top half of the medium to another well. If there are cell spheres in the new well, add the same amount of fresh medium.</li>
		<li>Change medium every the other day by repeating step 2.2.6.</li>
	</ol>
	</li>
	<li>Neural stem cell induction: 
	NOTE: Depending on cell conditions, about 5 or 7 days after transfection, monolayer adherent cells will reach 30 - 50% confluence (Figure&#160;1C).
	<ol>
		<li>Remove the supernatants containing floating cells and spheres and then add 1 ml of neural progenitor medium (Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12), containing 1x N2 supplement, 0.1% (w/v) bovine serum albumin (BSA), 1% (v/v) antibiotics, 20 ng/ml of basic fibroblast growth factor (bFGF), and 20 ng/ml of epidermal growth factor (EGF)) into the adherent cells.</li>
		<li>Optionally, dissociate the cell spheres by gently pipetting and centrifuge the cells at 170 x g for 10 min. Remove the supernatant and resuspend the cells in 1 ml of neural progenitor medium in a new 24-well plate coated with matrigel per instruction as back up.</li>
		<li>Change the medium every other day till the cells reach 60 - 80% confluent, usually after 1 week.</li>
		<li>Discard the supernatant and add in 1 ml of neural stem cell medium (serum free medium, NSC SFM). Dissociate the cells by using a cell scraper followed by very gentle pipetting.</li>
		<li>Remove the cells and seed all of them into one well of a 6-well plate. Add another 1 ml of neural stem cell medium to make 2 ml media for each well. Incubate the cells at 37 &#176;C in 5% CO2 incubator.</li>
		<li>Change medium every the other day.</li>
		<li>When cells reached 60% confluence, dissociate and replate the cells at 1:3 ratio following the steps in 2.3.4 and 2.3.6.</li>
	</ol>
	</li>
	<li>Freezing of neural stem cells:
	<ol>
		<li>Prepare neural stem cell freezing medium by adding 20% dimethyl sulfoxide (DMSO) into the neural stem cell medium. Keep the freezing medium on ice.</li>
		<li>Dissociate the cells in a 6-well plate, using cell scraper followed by gently pipetting. Count the cells. Centrifuge the cells at 170 x g for 10 min at RT. Discard the supernatant.</li>
		<li>Resuspend the cells in neural stem cell medium at 1 x 106/ml. Add the same volume of freezing medium to the cells.</li>
		<li>Add 1 ml (5 x 105 total cells) of cells in a cryovial. Freeze the cells using a Mr. Froster freezing container following the instruction.</li>
	</ol>
	</li>
</ol>
3. Neural Cell Differentiation
<ol>
	<li>Neuronal cells differentiation:
	<ol>
		<li>When neural stem cells reach 80% confluence, detach the neural stem cells using a rubber policeman and then dissociate by gently pipetting.</li>
		<li>Count the cells and adjust the concentration to 1 x 105/ml in neural stem cell medium.</li>
		<li>For neuronal differentiation, plate the cells on poly-D-lysine/laminin coated cover slips in 24-well plates at 1 x 105/well. Incubate the cells at 37 &#176;C in 5% CO2 incubator.</li>
		<li>Replace the medium with neural differentiation medium (DMEM/F12 containing 1x N2 supplement, 1x B27 supplement, 300 ng/ml cAMP, 0.2 mM vitamin C, 10 ng/ml brain derived neurotrophic factor (BDNF) and 10 ng/ml glial cell derived neurotrophic factor (GDNF)) after 24 hr. Incubate the cells at 37 &#176;C in 5% CO2 incubator.</li>
		<li>Change neural differentiation medium twice a week for 2 weeks.</li>
	</ol>
	</li>
	<li>Astroglial differentiation:
	<ol>
		<li>Dissociate the neural stem cells by pipetting.</li>
		<li>Count the cells and adjust the cell concentration to 5 x 104 cells/ml. Seed the cells on 24-well plates. Incubate the cells at 37 &#176;C in 5% CO2 incubator.</li>
		<li>Replace the medium with DMEM/F12 with 10% fetal bovine serum (FBS) and incubate the cells at 37 &#176;C in 5% CO2 Incubator.</li>
		<li>Change medium twice a week for 2 weeks. If cells reach confluence before 2 weeks, replate the cells following steps 3.2.1 and 3.2.2.</li>
	</ol>
	</li>
	<li>Oligodendrocyte differentiation:
	<ol>
		<li>Dissociate the neural stem cells by pipetting.</li>
		<li>Count the cells and seed 1 x 105 cells on poly-L-Ornithine coated 24-well plates in 1 ml of oligodendrocyte differentiation medium consisting of DMEM/F12 containing N2 supplement, 10 ng/ml of platelet derived growth factor-AA (PDGF-AA), 2 ng/ml of neurotrophin-3 (NT-3), 2 ng/ml of sonic hedgehog (Shh), and 3 nM of triiodothyronine (T3). Incubate the cells at 37 &#176;C in 5% CO2 incubator.</li>
		<li>Change medium twice a week for 2 weeks.</li>
		<li>Replace the medium with DMEM/F12 with 1xN2 supplement and 3 nM of T3 and culture the cells for an additional week for myelin basic protein (MBP) production.</li>
	</ol>
	</li>
</ol>
4. Immunofluorescence Staining
<ol>
	<li>Replace media with 4% paraformaldehyde (PFA). Incubate for 10 min at RT.</li>
	<li>Discard PFA and wash with PBS containing 0.1% Triton X-100 (PBS-T) 3 times, 5 min each time.</li>
	<li>Permeabilize cells with PBS containing 0.5% Triton X-100 for 10 min at RT.</li>
	<li>Block the cells with PBS-T containing 4% goat serum for 20 min at RT.</li>
	<li>Incubate the cells with corresponding primary antibodies diluted in PBS-T containing 0.1% BSA for 2 hr at RT or O/N in cold room.</li>
	<li>Wash the cells for 3 times with PBS-T, 5 min each time. Incubate the cells with corresponding secondary antibodies coupled with a fluorochrome using 1:400 dilution in PBS-T containing 0.1% BSA for 1 hr on a shaker in the dark.</li>
	<li>Wash the cells with PBS-T containing 4&#39;,6-diamidino-2-phenylindole (DAPI) for 10 min at RT.</li>
	<li>Wash the cells two more times with PBS-T for 5 min each at RT.</li>
	<li>Observe the labeled cells using a microscope and take photos (Figure 3).</li>
</ol>

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<li>Song, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+differentiation+of+patient+specific+iPS+cells+as+a+novel+approach+to+study+the+pathophysiology+of+multiple+sclerosis%5BTitle%5D)+AND+%22Stem+Cell+Research%22%5BJournal%5D)">Neural differentiation of patient specific iPS cells as a novel approach to study the pathophysiology of multiple sclerosis.</a> Stem Cell Research. 8 (2), 259-273 (2012).</li>
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</ol>

The study is funded by NIH intramural funds.

A detailed protocol to directly generate neural stem cells from peripheral hematopoietic progenitor cells is provided.
Compared to fibroblast cells, human peripheral blood is more accessible. Using the presented protocol, more than 1 x 105 hematopoietic progenitor cells, or CD34 positive cells, can be enriched from 10 ml of whole blood. Although contamination with platelets is usually not preventable, it can be easily reduced by lower speed centrifugation and it does not interfere w...

In vitro neuronal cultures have been used as a fundamental tool for studies of neurological diseases. Primary animal (mostly rodent) neural cultures1,2 and human neural cell lines derived from gliomas or other tumors are the most commonly used in such studies. However, it has been recognized that there are significant differences between rodent and human cells. Many findings based on rodents cannot be translated to humans. Furthermore, with the rapid developments in analyzing mass genomic information and the relatively easy gene editing and whole genome sequencing, the trend is more and more geared to discovering disease prone genes and delineating their functions and roles in specific diseases, which makes the few human neuronal cell lines have only limited usage. Theoretically, human primary neural cultures derived from samples of patient nervous system are the best choice but they are impossible to obtain; hence alternative methods are necessary. In recent years, some approaches have been pursued, with two being the most distinguishable. Following the development of the technique of generating induced pluripotent stem cells (iPSC) using mouse and human somatic cells3,4, neural cells could be further differentiated from them5-7. However, generating and characterizing iPSC demands intensive labor, techniques, and time input, sometimes even prohibitively. Shortly after, another approach was developed to directly transform neuronal cells from somatic cells8,9. As the resulting neurons are non-proliferative, it limits its application in intensive studies and drug screening, which requires a large amount of cells. To take advantages of both techniques, direct derivation of neural stem/progenitor cells from somatic cells has been explored by several groups10-12, which bypasses the tedious process of iPSC generation and characterization but still provides a decent number of neural stem cells for later neural differentiation. We have previously shown that following the introduction of the Yamanaka transcription factors into hematopoietic progenitor cells, neural stem cells could be directly generated using a neural progenitor cell selecting medium13. Here, we report the method in detail.

<table><tbody><tr><td>Lymphocyte separation medium</td><td>Lonza</td><td>17-829E</td><td>1x</td></tr><tr><td>SepMate</td><td>Stemcell Technologies</td><td>15415</td><td>15 ml</td></tr><tr><td>Human serum</td><td>Invitrogen</td><td>34005-100</td><td></td></tr><tr><td>Antibiotics</td><td>Gibco</td><td>15240-062</td><td>1%</td></tr><tr><td>CD34 MultiSort Kit</td><td>Miltenyi Biotec</td><td>130-056-701</td><td></td></tr><tr><td>EDTA</td><td>Cellgro</td><td>46-034-Cl</td><td>2 mM</td></tr><tr><td>MACS LS column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>StemSpan SFEM medium</td><td>Stemcell Technologies</td><td>9650</td><td>1x</td></tr><tr><td>IMDM</td><td>Quality Biologicals</td><td>112-035-101</td><td>1x</td></tr><tr><td>TPO</td><td>Peprotech</td><td>300-18</td><td>100 ng/ml</td></tr><tr><td>Flt-3</td><td>Peprotech</td><td>300-19</td><td>100 ng/ml</td></tr><tr><td>SCF</td><td>Peprotech</td><td>300-07</td><td>100 ng/ml</td></tr><tr><td>IL-6</td><td>Peprotech</td><td>200-06</td><td>20 ng/ml</td></tr><tr><td>IL-7</td><td>Peprotech</td><td>200-07</td><td>20 ng/ml</td></tr><tr><td>IPS Sendai Reprogramming Kit</td><td>Life Technologies</td><td>A1378001</td><td></td></tr><tr><td>Cell scraper</td><td>Sarstedt</td><td>83.183</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>CryoTube vials</td><td>Thermo</td><td>368632</td><td></td></tr><tr><td>Mr. Frosty container</td><td>Thermo</td><td>5100-0001</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>12400-024</td><td>1x</td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502-048</td><td>1x</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2934</td><td>0.1% (w/v)</td></tr><tr><td>bFGF</td><td>Peprotech</td><td>100-18B</td><td>20 ng/ml</td></tr><tr><td>EGF</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>17504-044</td><td>1x</td></tr><tr><td>NSC serum free medium</td><td>Life Technologies</td><td>A1050901</td><td>1x</td></tr><tr><td>Poly-D-lysine/laminin coated cover slips</td><td>BD Bioscences</td><td>354087</td><td></td></tr><tr><td>cAMP</td><td>Sigma</td><td>A9501</td><td>300 ng/ml</td></tr><tr><td>Vitamin C</td><td>Sigma</td><td>A0278</td><td>0.2 mM</td></tr><tr><td>BDNF</td><td>Peprotech</td><td>450-02</td><td>10 ng/ml</td></tr><tr><td>GDNF</td><td>Peprotech</td><td>450-10</td><td>10 ng/ml</td></tr><tr><td>Poly-L-ornithine</td><td>Sigma</td><td>P4957</td><td>1x</td></tr><tr><td>PDGF-AA</td><td>Peprotech</td><td>100-13A</td><td>10 ng/ml</td></tr><tr><td>NT-3</td><td>Peprotech</td><td>450-03</td><td>2 ng/ml</td></tr><tr><td>Shh</td><td>Peprotech</td><td>1314-SH/CF</td><td>2 ng/ml</td></tr><tr><td>T3</td><td>Sigma</td><td>T6397</td><td>3 nM</td></tr><tr><td>PFA</td><td>Sigma</td><td>P6148</td><td>4%</td></tr><tr><td>PBS</td><td>Quality Biological</td><td>119-069-101</td><td>1x</td></tr><tr><td>Goat serum</td><td>Sigma</td><td>G9023</td><td>4%</td></tr><tr><td>TritonX-100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Mouse monoclonal anti-Nestin</td><td>Millipore</td><td>AB5922</td><td>1:1,000 dilution</td></tr><tr><td>Anti-SOX2 antibody</td><td>Applied Stemcell</td><td>ASA0120</td><td>Ready to use</td></tr><tr><td>Mouse anti-&amp;beta;III-tubulin antibody</td><td>Promega</td><td>G712A</td><td>1:1,000 dilution</td></tr><tr><td>Rabbit anti-GFAP antibody</td><td>Sigma</td><td>G4546</td><td>1:100 dilution</td></tr><tr><td>Anti-O4 antibody</td><td>R&amp;amp;D Systems</td><td>MAB1326</td><td>IgM; 1 ng/ml</td></tr><tr><td>Alexa Fluor 594 goat anti-rabbit antibody</td><td>Life techniologies</td><td>A11012</td><td>1:400 dilution</td></tr><tr><td>Alexa Fluor 488 goat anti-mouse antibody</td><td>Life techniologies</td><td>A11001</td><td>1:400 dilution</td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgM antibody</td><td>Life techniologies</td><td>A21042</td><td>1:250 dilution</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>1 &amp;mu;g/ml</td></tr></tbody></table>

direct induction of human neural stem cells from peripheral blood hematopoietic progenitor cells

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

The quality of CD34 cells is critical for the success of neural stem cell transduction. High quality CD34 cells proliferate during the first couple of days of culture and appear as non adherent, homogenously round cells, floating just above the bottom of culture vessels (Figure 1A). The successful infection results in cell aggregates, which expands over time (Figure 1B), while non specific cell aggregates may occur during cell culture, that expand and the associations are loose. Adherent...

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

The overall goal of this procedure is to directly induce neural stem cells from blood cells. This is accomplished by first enriching CD 34 positive hematopoietic progenitor cells from blood. The second step is to transfect CD 34 cells with sendi virus containing transcriptional factors.Next, the neural stem cells are derived using selective medium. The final step is to differentiate the induced neural stem cells into neurons, astroglia, and oligodendrocytes. Ultimately, immunofluorescence microscopy is used to characterize the resulting cells.The main advantage of this technique over existing method, such as duration of neurons directly from fibroblast, also IPS generation, is that this method bypasses the tedious process of IPS generation and calculation, but still provide a large amount of neuronal cells which can relate, differentiate to neurons. I first had the idea for this method when I noticed that attached transfected CT 34 cells actually shows very similar to primary cultured neuro progenic cells demonstrating the procedure will be Marines, a technician from my life. After isolating CD 34 cells from peripheral blood mononuclear cells according to instructions in the text protocol, begin the process of culturing the CD 34 cells by Resus, suspending the CD 34 cells in CD 34 media then seed the cells into a 24 well plate at three times 10 to the fifth cells per well in one milliliter of medium.After incubating for 24 hours in a 37 degree Celsius tissue culture incubator with 5%carbon dioxide, collect the floating cells and centrifuge the cell suspension at 110 times G at room temperature. Then discard the supernatant and resuspend the cells in fresh CD 34 maintenance medium. See a new 24 well plate at one times 10 to the fifth cells per well in one milliliter of medium, incubate at 37 degrees Celsius in a 5%carbon dioxide incubator for five additional days, replacing half of the medium every other day.By carefully aspirating the top half of supernatant while the CD 34 cells are on the bottom of the wells as shown here on the fifth day. After plating, collect CD 34 cells and centrifuge the cells at 170 times G for 10 minutes at room temperature, discard the supernatant, resuspend the cells as before and seed a fresh 24 well plate at one times 10 to the fifth cells per well in one milliliter of media. Next thaw a cyto tune IPS Sendi reprogramming kit.By first immersing the bottoms of the tubes in a 37 degrees Celsius water bath for 10 seconds and then completely thaw at room temperature. Briefly centrifuge the tubes and put them on ice. Prepare the Sendai virus mixture by mixing the recommended volume of each virus listed on the certificate of analysis for the corresponding lot.A multiplicity of infection of 15 is recommended. Add the Sendai virus mix to each well of CD 34 cells. Then mix the wells by gently shaking the plate.Incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator after 24 hours. Observe the transduction efficiency. Notice that cell aggregates and spheres such as those shown here are indicators of successful transduction.Then incubate the cells in the tissue culture incubator for five to seven days until attached cells appear and reach 30 to 50%confluence as shown here every other day. Change half of the medium by carefully transferring the top half of the medium to another well. If cell spheres are transferred to the new, well add an equal amount of fresh medium.Check the co fluency of the cells using the inverted microscope. When the cells are 30 to 50%confluent, aspirate the supernatant and reserve and then add one milliliter of neural progenitor medium to each well to make a backup plate using the spheres in the supernatant centrifuge, the reserve supernatant at 170 times G for 10 minutes. Then aspirate the supernatant and resuspend the pellet in neural progenitor medium plate in a matrigel coated 24 well plate.As before, change the medium in each plate of cells every other day until the cells reach 60 to 80%confluence. Usually after one week. Once the cells reach the required confluence, aspirate the supernatant and add one milliliter of neural stem cell medium to each.Well then dissociate the cells by using a cell scraper, followed by very gentle pipetting. Next, transfer the cells from one well of the 24 well plate into one well of a six. Well plate.Add another milliliter of medium to the well and after repeating the procedure for all wells, place the six well plate into the 37 degrees Celsius 5%carbon dioxide incubator. Change the full volume of medium every other day until the cells reach 60%confluence after which time dissociate and reflate the cells at a one to three ratio as just shown to freeze a portion of undifferentiated neural stem cells. First, prepare the stem cell freezing medium by adding 20%dimethyl sulf oxide or DMSO to the neural stem cell medium and place on ice.Then dissociate the cells in the six well plate. Using a cell scraper and gentle pipetting. Reserve an aliquot of the suspension for a cell count and then centrifuge the cells at 170 times G for 10 minutes at room temperature while the cells are spinning, count the cells in the aliquot of suspension using a hemo, cytometer or automated cell counter and calculate the volume of medium required to give a cell concentration of one times 10 to the six cells per milliliter.After the spin has finished, discard the supernatant and resuspend the pellet in neural stem cell medium at one times 10 to the six cells per milliliter. Then add an equal volume of freezing medium pipette five times 10 to the fifth cells in a volume of one milliliter into a cryo vial and freeze the cells in a freezing container according to the manufacturer's instructions. When the neural stem cells in the six well plate reach 80%confluence, detach them using a rubber policeman and then dissociate by gentle pipetting.Pool the cell suspension into a sterile tube. Count the cells using a hemo, cytometer or automated cell counter and adjust the concentration of the cell suspension to one times 10 to the fifth cells per milliliter with neural stem cell medium. Then pipette one milliliter of the cell suspension onto a poly D lysine and laminate coated cover slip in one well of a 24 well plate after repeating the procedure for the desired number of wells.Incubate as before for 24 hours. After the incubation time has elapsed, aspirate the media and replace with neural differentiation. Medium incubate for two weeks at 37 degrees Celsius and 5%carbon dioxide changing the medium twice a week after two weeks.The neuronal stem cells should be fully differentiated as shown here and ready for downstream applications such as immunofluorescence staining. The first two images show that neural stem cells express nest in and SOX two. While the third image demonstrates that OCT four is not expressed, DPI is used as a nuclear marker and the scale bar represents 200 microns.After following the differentiation protocol outlined in the video, the differentiated neurons express the neuronal marker beta three tubulin. The human neural stem cells can also be differentiated into glial cells. When differentiated into astrocytes, the cells express the astrocytic marker GFAP.Finally, in this image, the cells have been differentiated into oligodendrocytes and express the oligodendrocyte marker. O four. Once mastered the generation of induced human neural stem cells from peripheral blood can be done in approximately three weeks.If performed properly, The neuro stem cells could be further differentiated neurons and unclear the method by is the lengthy and complicated IPS generation processes and could be a easy option to generate patient specific neuronal cultures after its development. This technique path way for researchers in the field of neuroscience to explore individual cell culture modeling for various neurological disorders, which might better represent the genetic differences in specific disorders, especially from individual patient samples.

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Research

JoVE Journal

Developmental Biology

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Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

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Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

This video demonstrates the reprogramming of peripheral blood mononuclear cells (PBMNCs) into induced neural stem cells (iNSCs) using Sendai virus. Sendai virus is a non-integrating virus that offers an efficient and safe vehicle for reprogramming cells.

Generation of Induced Neural Stem Cells (iNSCs) from Peripheral Blood Mononuclear Cells (PBMNCs): A Sendai Virus-based Procedure to Reprogram PBMNCs into iNSCs

This video demonstrates the direct reprogramming of genetically modified hematopoietic progenitor cells into neuronal stem cells. Transcription factors expressed by hematopoietic progenitor cells trigger a cascade of molecular events facilitating their transformation. Further specific media is used to select and grow induced neuronal stem cells.

Direct Reprogramming of Hematopoietic Progenitor Cells into Neural Stem Cells

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

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Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson's disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Learn to directly induce human neural stem cells from peripheral blood CD34+ hematopoietic progenitors using Sendai virus transfection and selective medium. This method bypasses iPS generation for patient-specific neural cultures.

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

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