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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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 border="0" cellpadding="0" cellspacing="0" width="584">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Material List</td>
			<td style="width:171px;"></td>
			<td style="width:135px;"></td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Name</td>
			<td style="width:171px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:72px;">Comments</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Lymphocyte Separation Medium</td>
			<td style="width:171px;">Lonza</td>
			<td style="width:135px;">17-829E</td>
			<td style="width:72px;">1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">SepMate</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td align="right" style="width:135px;">15415</td>
			<td style="width:72px;">15 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Human Serum</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:135px;">34005-100</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Antibiotics</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:135px;">15240-062</td>
			<td align="right" style="width:72px;">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">CD34 MultiSort Kit</td>
			<td style="width:171px;">Miltenyi Biotec</td>
			<td style="width:135px;">130-056-701</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">EDTA</td>
			<td style="width:171px;">Cellgro</td>
			<td style="width:135px;">46-034-Cl&#160;</td>
			<td style="width:72px;">2mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">MACS LS Column</td>
			<td style="width:171px;">Miltenyi Biotec</td>
			<td style="width:135px;">130-042-401</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">StemSpan SFEM Medium</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td align="right" style="width:135px;">9650</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IMDM</td>
			<td style="width:171px;">Quality Biologicals</td>
			<td style="width:135px;">112-035-101</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">TPO</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">300-18</td>
			<td style="width:72px;">100 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Flt-3</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">300-19</td>
			<td style="width:72px;">100 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">SCF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">300-07</td>
			<td style="width:72px;">100 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IL-6</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">200-06</td>
			<td style="width:72px;">20 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IL-7&#160;</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">200-07</td>
			<td style="width:72px;">20 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">IPS Sendai Reprogramming Kit</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">A1378001</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Cell scraper</td>
			<td style="width:171px;">Sarstedt</td>
			<td align="right" style="width:135px;">83.183</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">DMSO</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">D2650</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">CryoTube vials</td>
			<td style="width:171px;">Thermo</td>
			<td align="right" style="width:135px;">368632</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Mr. Frosty container</td>
			<td style="width:171px;">Thermo</td>
			<td style="width:135px;">5100-0001</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">DMEM/F12</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">12400-024</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">N2 supplement</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">17502-048</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Bovine serum albumin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">A2934</td>
			<td style="width:72px;">0.1% (w/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">bFGF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">100-18B</td>
			<td style="width:72px;">20 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">EGF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">AF-100-15</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">B27 supplement</td>
			<td style="width:171px;">Life technologies</td>
			<td style="width:135px;">17504-044</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">NSC serum free medium</td>
			<td style="width:171px;">Life Technologies</td>
			<td style="width:135px;">A1050901</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="40">
			<td height="43" rowspan="2" style="height:43px;width:208px;">Poly-D-lysine/laminin coated cover slips</td>
			<td rowspan="2" style="width:171px;">BD Bioscences</td>
			<td align="right" rowspan="2" style="width:135px;">354087</td>
			<td rowspan="2" style="width:72px;"></td>
		</tr>
		<tr height="3">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">cAMP</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">A9501</td>
			<td style="width:72px;">300 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Vitamin C</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">A0278</td>
			<td style="width:72px;">0.2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">BDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">450-02</td>
			<td style="width:72px;">10 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">GDNF</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">450-10</td>
			<td style="width:72px;">10 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Poly-L-Ornithine</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">P4957</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PDGF-AA</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">100-13A</td>
			<td style="width:72px;">10 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">NT-3</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">450-03</td>
			<td style="width:72px;">2 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Shh</td>
			<td style="width:171px;">Peprotech</td>
			<td style="width:135px;">1314-SH/CF</td>
			<td style="width:72px;">2 ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">T3</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">T6397</td>
			<td style="width:72px;">3 nM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PFA</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">P6148</td>
			<td align="right" style="width:72px;">4%</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:208px;">PBS</td>
			<td style="width:171px;">Quality Biological</td>
			<td style="width:135px;">119-069-101</td>
			<td style="width:72px;">1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Goat serum</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">G9023</td>
			<td align="right" style="width:72px;">4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">TritonX-100</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">T9284</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Mouse monoclonal anti-Nestin</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:135px;">AB5922</td>
			<td style="width:72px;">1:1000 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Anti-SOX2&#160; antibody</td>
			<td style="width:171px;">Applied Stemcell</td>
			<td style="width:135px;">ASA0120</td>
			<td style="width:72px;">Ready to use</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Mouse anti-&#946;III-tubulin antibody</td>
			<td style="width:171px;">Promega</td>
			<td style="width:135px;">G712A</td>
			<td style="width:72px;">1:1000 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:208px;">Rabbit anti-GFAP antibody</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">G4546</td>
			<td style="width:72px;">1:100 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Anti-O4 antibody</td>
			<td style="width:171px;">R&#38;D Systems</td>
			<td style="width:135px;">MAB1326</td>
			<td style="width:72px;">IgM; 1 ng/ml</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:208px;">Alexa Fluor 594 goat anti-rabbit antibody</td>
			<td style="width:171px;">Life techniologies</td>
			<td style="width:135px;">A11012</td>
			<td style="width:72px;">1:400 dilution</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:208px;">Alexa Fluor 488 goat anti-mouse antibody</td>
			<td style="width:171px;">Life techniologies</td>
			<td style="width:135px;">A11001</td>
			<td style="width:72px;">1:400 dilution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:208px;">Alexa Fluor 488 goat anti-mouse IgM antibody</td>
			<td style="width:171px;">Life techniologies</td>
			<td style="width:135px;">A21042</td>
			<td style="width:72px;">1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">DAPI</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:135px;">D9542</td>
			<td style="width:72px;">1 ug/ml</td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells at JoVE.com

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

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 ...

direct-induction-human-neural-stem-cells-from-peripheral-blood

Research

JoVE Journal

Developmental Biology

12.4K 次观看.  National Institutes of Health. 的方法的开发是为了直接从外周血细胞富集的造血祖细胞衍生的人神经干细胞。 

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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

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

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

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

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

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.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...