Name: Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells (Video) | JoVE Journal
Uploaded: 2017-06-14T13:00:00.000+00:00
Duration: 10 min 16 s
Description: Learn hypoxic preconditioning of marrow-derived progenitor cells to generate mature Schwann cells for regenerative medicine. This method enables autologous cell therapy for nerve injury repair and remyelination.

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5 minutes

The authors would like to acknowledge Dr. Nai-Sum Wong for providing the hypoxia chamber apparatus and Ms. Alice Lui for the technical support.

All procedures involving animals were performed in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Committee on Use of Live Animals for Teaching and Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong. Human bone marrow samples were obtained from the iliac crest of healthy donors after obtaining informed consent. Protocols were approved by the Institutional Review Board, The University of Hong Kong.
1. Preparation of Rat MSC Cultures
<ol>
	<li>Harvest of MSCs from the femur
	<ol>
		<li>Autoclave all dissection tools (i.e., fine dissecting scissors, blunt-tipped cutting scissors, and toothed forceps) at 180 &#176;C for at least 2 h prior to use.</li>
		<li>Prepare MSC growth medium comprising of minimal essential medium - alpha modification (&#945;MEM) supplemented with 15% fetal bovine serum (FBS) and penicillin/streptomycin (P/S, 1% v/v).</li>
		<li>Sacrifice young male Sprague Dawley rats (200-250 g bodyweight) by pentobarbitone overdose (240 mg/kg bodyweight, intraperitoneal). 
		NOTE: Marrow samples from different rats should be processed separately.</li>
		<li>Place the sacrificed animals in supine position. Clean their abdomen and lower limbs thoroughly with 70% ethanol.</li>
		<li>Remove the skin and subcutaneous tissue over the medial thighs using fine dissecting scissors and forceps. Remove the thigh muscles circumferentially until the femur is exposed. Continue this proximally and distally until the knee and hip joints are seen. Disarticulate the femur through the hip and knee joint using blunt-tipped cutting scissors. 
		NOTE: Do not transect the femur to expose the marrow cavity at this stage. Transfer intact femurs to a laminar flow tissue culture hood for further processing.</li>
		<li>Use blunt tipped cutting scissors to transect the distal and proximal ends of the femur through the metaphysis.</li>
		<li>Place a 70 &#181;m cell strainer over a 50 mL conical tube. Insert a 21 G, 10 mL syringe containing phosphate-buffered saline (PBS, 10 mM Na2HPO4, pH 7.4) into the exposed femoral canal and flush the marrow content into the conical tube by repeated flushing. 
		NOTE: Approximately 20 mL of PBS is used to flush each femur. If the color of the flushed content remains blood-stained and turbid, a larger volume can be used.</li>
		<li>Collect the cells by centrifugation at 480 x g for 5 min. Discard the supernatant. Resuspend the cell pellet in 10 mL of MSC growth medium. Plate the cells onto a 10 cm tissue culture dish.Place the tissue culture dishes into a cell incubator (37 &#176;C, 5% CO2). Record the initial day of plating as day 0. 
		NOTE: In all steps involving centrifugation, set the brake for maximal deceleration.</li>
	</ol>
	</li>
	<li>Establishment and expansion of MSC colonies 
	NOTE: This protocol relies on tissue culture plastic adherence as a means to select for MSCs from within the marrow cavity11. The bone marrow content is allowed to adhere to the tissue culture plastic for 2 days.
	<ol>
		<li>On day 2, rinse the culture plates three times with 10 mL of PBS to remove non-adherent cells. Replace the PBS with 10 mL of MSC growth medium after rinsing. Wash the cells with PBS and replenish with MSC growth medium every 3 days. 
		NOTE: MSC colonies should be visible by day 6-7 (Figure 2A).</li>
		<li>Passage the cells by day 10 by removing the growth medium and rinsing the cells with PBS. Add 1.5 mL of recombinant enzymatic cell dissociation reagent and incubate at 37 &#176;C for 5 min. Add 3 mL of MSC growth medium to neutralize the reaction. Collect the detached cells by centrifugation at 250 x g for 5 min.</li>
		<li>Quantify the cells within the pellet using a hemocytometer after an appropriate dilution in PBS.</li>
		<li>Seed passaged cells at a density of 40,000 cells/cm2 into a 10-cm culture plate in MSC growth medium. 
		NOTE: Rat MSCs should reach 80-90% confluence within 2 days of passaging (Figure 2B). Cells can be passaged as described in step 1.2.2 for up to 8 passages. MSCs can be characterized by means of immunocytochemistry and their capacity for trilineage differentiation (Figure 3)12. Only MSC cultures between passages 3 and 8 are subject to subsequent hypoxic preconditioning and neural progenitor enrichment. MSCs of greater passage number adopt a flattened morphology (Figure 2C) and do not produce sufficient numbers of neural progenitors. These cultures should be discarded.</li>
	</ol>
	</li>
</ol>
2. Preparation of Human BMSC Cultures
<ol>
	<li>Dilute 1 mL of human bone marrow aspirate with 9 mL of MSC growth medium and plate the cells on a 10-cm tissue culture dish. Maintain the cultures in a cell incubator (37 &#176;C, 5% CO2).</li>
	<li>Remove the medium after 2 days and gently rinse the cultures three times with 10 mL of PBS to remove non-adherent cells. After the final rinse, remove the PBS and replace it with 10 mL of MSC growth medium. Replenish the growth medium after every 3 days of culture after the PBS rinse. 
	NOTE: MSC colonies should be visible by day 6-7. The number of colonies may vary between subjects.</li>
	<li>Passage the cells on day 10, as described in step 1.2.2. Quantify the cells within the pellet using a hemocytometer after an appropriate dilution in PBS. Seed the passaged cells at a density of 40,000 cells/cm2 onto a 10 cm culture plate in MSC growth medium. 
	NOTE: Human MSCs (Figure 2D) demonstrate a similar morphology to rat MSCs and likewise should reach 80-90% confluence within 2 days of passaging. They should be characterized by means of immunocytochemistry and their capacity for trilineage differentiation12. As with rat MSCs, human MSCs that are between passage 3 and 8 are subject to subsequent hypoxic preconditioning and neural progenitor enrichment.</li>
</ol>
3. Hypoxic Preconditioning
<ol>
	<li>Disassemble the hypoxia chamber components (i.e., base, lid, trays) after releasing the ring clamp and wipe the individual parts clean with 70% ethanol. Place the chamber components within a laminar flow tissue culture hood for sterilization under UV light for 15 min.</li>
	<li>Prior to hypoxic preconditioning, remove the medium and rinse the rat and human MSC cultures (sections 1 and 2) with 10 mL of PBS. Replace the PBS with 10 mL of MSC growth medium supplemented with 25 mM HEPES. 
	NOTE: The MSCs cultured upon 10 cm dishes should have reached 80-90% confluency prior to be being subject to hypoxic preconditioning.</li>
	<li>Place the culture dishes within the hypoxia chamber. Reassemble the chamber components and tighten the ring clamp. Flush a gas mixture of 99% N2/1% O2 into the chamber at a flow rate of 10 L/min for 5 min.</li>
	<li>Seal the connecting ends of the hypoxia chamber to ensure there is no gas leakage. Place the chamber inside the cell incubator (37 &#176;C, 5% CO2) for 16 h.</li>
	<li>Upon completion of the hypoxic preconditioning, remove the cultures from the chamber in preparation for the subsequent neural progenitor enrichment culture.</li>
</ol>
4. Neural Progenitor Enrichment Culture
<ol>
	<li>Prepare neural progenitor medium comprised of Dulbecco&#39;s modified eagle medium/Ham&#39;s nutrient mixture F12 (DMEM/F12) supplemented with B27 (2% v/v), basic fibroblast growth factor (bFGF, 20 ng/mL), epidermal growth factor (EGF, 20 ng/mL), and P/S (1% v/v).</li>
	<li>Detach the hypoxic preconditioned rat/human MSCs, as described in step 1.2.2. Collect the detached cells by centrifugation at 250 x g for 5 min. Quantify the cells within the pellet using a hemocytometer after appropriate dilution in PBS.</li>
	<li>Resuspend the cells in neural progenitor medium and seed on low-attachment, 6-well plates at a density of 6,000 cells/cm2. Place the culture in a cell incubator (37 &#176;C, 5% CO2) for 12 days. Replenish 75% of the neural progenitor medium every 3 days. 
	NOTE: Sizeable non-adherent cell clusters should be observed by day 6-7. By day 10-12, neurospheres with a diameter &#8805; 100 &#181;m can be observed (Figure 4). Hypoxic preconditioned MSCs should yield more neurospheres as compared to MSCs cultured under normoxic conditions9.</li>
	<li>Collect neurospheres on day 12 by aspirating them into a 10-mL pipette and transferring them to a 15 mL conical tube. Centrifuge the neurospheres at 250 x g for 5 min. 
	NOTE: Neurospheres can be characterized on day 12 for neural progenitor markers, such as nestin and GFAP7.</li>
</ol>
5. Generation of Fate-committed Schwann Cells via Coculture with DRG Neurons
<ol>
	<li>Preparation of purified rat DRG neurons
	<ol>
		<li>Autoclave all dissection tools (i.e., dissection scissors, forceps, two microdissection forceps, and microdissecting scissors) at 180 &#176;C for at least 2 h prior to use.</li>
		<li>Coat 6-well tissue culture plates with poly-D-lysine (PDL, 10 &#181;g/mL in PBS) at 4 &#176;C overnight. Remove the PDL and rinse with 1.5 mL of PBS per well.</li>
		<li>Proceed with coating the plates with laminin (10 &#181;g/mL in PBS) at 37 &#176;C for 2 h. Rinse the plates with 1.5 mL of PBS per well.</li>
		<li>Prepare DRG neuron maintenance medium, comprised of neurobasal medium supplemented with B27 (2% v/v), L-glutamine (1% v/v), nerve growth factor (NGF, 20 ng/mL), and P/S (1% v/v).</li>
		<li>Prepare DRG neuron purification medium, comprised of neurobasal medium supplemented with B27 (2% v/v), L-glutamine (1%), NGF (20 ng/mL), P/S (1%), fluorodeoxyuridine (FDU, 10 &#181;g/mL), and uridine (10 &#181;g/mL).</li>
		<li>Sacrifice pregnant rats at gestational day 14-15 by pentobarbital overdose (240 mg/kg bodyweight, intraperitoneal).</li>
		<li>Place the sacrificed animals in supine position. Clean their abdomen thoroughly with 70% ethanol.</li>
		<li>Cut the lower abdominal wall of the animal longitudinally using fine dissecting scissors and forceps. Identify and remove the uterus using dissection scissors. Cut the uterine wall to expose and extract the embryos. Transfer the embryos to a sterile, 10 cm culture dish filled with PBS. Place the culture dish on ice.</li>
		<li>Transfer the embryo intended for dissection to a sterile, 10 cm culture dish filled with PBS (room temperature) and position it beneath a dissection microscope. Have the embryo in prone position. 
		NOTE: The whitish spinal cord and the attached DRGs can be seen over the dorsal aspect of the embryo, through its translucent skin.</li>
		<li>Insert microdissecting forceps along either side of the spinal cord and use blunt dissection to begin separating the spinal cord from the surrounding soft tissue. Cut the spinal cord free from the animal using microdissecting forceps along the neck opening and tail stub. Perform further blunt dissection over the ventral aspect of the cord to free it from surrounding soft tissue.</li>
		<li>Use microdissecting forceps to remove residual soft tissue over the dorsal aspect of the freed spinal cord. 
		NOTE: At this stage, only the spinal cord, nerve roots, and attached DRG should remain.</li>
		<li>Detach individual DRGs from their connecting nerve roots using microdissecting forceps. Use a pipette pen attached to a 1 mL tip to transfer the DRGs to a 1.5 mL, sterile centrifuge tube containing PBS. 
		NOTE: For each 1.5 mL tube, a maximum of 100 DRGs can be accommodated.</li>
		<li>Centrifuge the DRGs at 250 x g for 5 min and resuspend them in recombinant enzymatic cell dissociation reagent (200 &#181;L per tube). Incubate (37 &#176;C, 5% CO2) for 10 min. Centrifuge the DRGs at 250 x g for 5 min, remove the supernatant, and resuspend in DRG neuron maintenance medium. Dissociate the pellet by gentle trituration using a 200 &#181;L pipette tip. Quantify the cells within the pellet using a hemocytometer after an appropriate dilution.</li>
		<li>Seed the cells at a density of 5,000 cells/cm2 into PDL/laminin-coated 6-well plates in 1.5 mL of DRG neuron maintenance medium per well. After two days of culture, remove the DRG neuron maintenance medium, rinse with PBS, and replace with DRG neuron purification medium. 
		NOTE: For each purification cycle, treat the DRG cultures with purification medium for 2 days, followed by 1 day of incubation in maintenance medium. After 3-4 purification cycles, the removal of all endogenous glia is expected7. This should take approximately 14 days. Purified cultures test positive for the neuronal marker TUJ1 and are absent for S100&#946; expression (Figure 5).</li>
	</ol>
	</li>
	<li>Generation of Schwann cell-like cells
	<ol>
		<li>Prepare glial induction medium comprised of &#945;MEM supplemented with &#946;-Heregulin (100 ng/mL), bFGF (10 ng/ml), platelet-derived growth factor (PDGF-AA, 5 ng/mL), FBS (10%), and P/S (1% v/v).</li>
		<li>Plate the neurospheres prepared in section 4 in PDL/laminin-coated 6-well plates at a density of 5-10 spheres per cm2 in 1.5 mL of glial induction medium per well. Replace glial induction medium every 2 days after rinsing the cells with PBS. 
		NOTE: Cells from seeded neurospheres are seen to migrate outwards by day 2. By day 7, migratory cells have a tapered appearance and should demonstrate immunopositivity for the Schwann cell markers p75 neurotrophin receptor (p75) and S100&#946;7. These cells are referred to as SCLCs.</li>
	</ol>
	</li>
	<li>Coculture of SCLCs with DRG neurons
	<ol>
		<li>Prepare coculture medium comprised of DRG neuron maintenance medium (step 5.1.4) and glial induction medium (step 5.2.1) at a 1:1 volume-to-volume ratio.</li>
		<li>Prepare Schwann cell maintenance medium comprised of DMEM/F12 supplemented with FBS (5%), &#946;-Heregulin (10 ng/mL), and P/S (1% v/v).</li>
		<li>Remove the culture medium from day 7 SCLCs, rinse them with PBS, and incubate them with 0.5 mL/well of recombinant enzymatic cell dissociation reagent at 37 &#176;C for 5 min. Resuspend the SCLCs in coculture medium.</li>
		<li>Quantify the cells using a hemocytometer after an appropriate dilution.</li>
		<li>Seed the SCLCs onto purified DRG neuron cultures at a density of 1,000 cells/cm2. Maintain the cocultures for 14 days, with medium replacement every 2 days. 
		NOTE: During coculture, SCLCs acquired the spindle-like morphology that is typical of mature Schwann cells (Figure 6). These cells persist in their phenotype after the withdrawal of growth factors and are able to myelinate axons in vitro and in vivo7,10. The positivity of Schwann cell markers (i.e., p75 and S100&#946;) should be monitored by immunofluorescence.</li>
		<li>Upon completion of coculture, passage fate-committed Schwann cells, as described in step 1.2.2. Use 0.5 mL of dissociation reagent per well. Quantify the cells using a hemocytometer after an appropriate dilution.</li>
		<li>Resuspend fate-committed Schwann cells in Schwann cell maintenance medium at a density of 10,000 cells/cm2. Seed cells into PDL/laminin-coated 6-well plates for immunofluorescence. 
		NOTE: Cocultures inevitably contain MSCs that that have adopted a fibroblast cell fate7. These cells are passaged together with fate-committed Schwann cells upon the completion of the coculture. Schwann cells lying on top of this fibroblast substratum can be easily detached and further expanded following rinsing with &#34;cold jets&#34; of PBS (4 &#176;C), as has been described elsewhere13. Fate-committed Schwann cells can be expanded in maintenance medium for 1 month.</li>
	</ol>
	</li>
</ol>

<ol><li>Wiliams, R. R., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Schwann+cell+transplantation%3A+a+repair+strategy+for+spinal+cord+injury%3F%5BTitle%5D)+AND+%22Prog+Brain+Res%22%5BJournal%5D)">Schwann cell transplantation: a repair strategy for spinal cord injury?</a> Prog Brain Res. 201, 295-312 (2012).</li>
<li>Kanno, H., Pearse, D. D., Ozawa, H., Itoi, E., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Schwann+cell+transplantation+for+spinal+cord+injury+repair%3A+its+significant+therapeutic+potential+and+prospectus%5BTitle%5D)+AND+%22Rev+Neurosci%22%5BJournal%5D)">Schwann cell transplantation for spinal cord injury repair: its significant therapeutic potential and prospectus.</a> Rev Neurosci. 26 (2), 121-128 (2015).</li>
<li>Lindvall, O., Kokaia, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cells+in+human+neurodegenerative+disorders--time+for+clinical+translation%3F%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Stem cells in human neurodegenerative disorders--time for clinical translation?</a> J Clin Invest. 120 (1), 29-40 (2010).</li>
<li>Terzic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+Differentiation+of+Oligodendrocyte+Progenitor+Cells+From+Mouse+Induced+Pluripotent+Stem+Cells%5BTitle%5D)+AND+%22Cell+Transplant%22%5BJournal%5D)">Directed Differentiation of Oligodendrocyte Progenitor Cells From Mouse Induced Pluripotent Stem Cells.</a> Cell Transplant. 25 (2), 411-424 (2016).</li>
<li>Takashima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroepithelial+cells+supply+an+initial+transient+wave+of+MSC+differentiation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Neuroepithelial cells supply an initial transient wave of MSC differentiation.</a> Cell. 129 (7), 1377-1388 (2007).</li>
<li>Felling, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+stem%2Fprogenitor+cells+participate+in+the+regenerative+response+to+perinatal+hypoxia%2Fischemia%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Neural stem/progenitor cells participate in the regenerative response to perinatal hypoxia/ischemia.</a> J Neurosci. 26 (16), 4359-4369 (2006).</li>
<li>Shea, G. K., Tsui, A. Y., Chan, Y. S., Shum, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone+marrow-derived+Schwann+cells+achieve+fate+commitment--a+prerequisite+for+remyelination+therapy%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Bone marrow-derived Schwann cells achieve fate commitment--a prerequisite for remyelination therapy.</a> Exp Neurol. 224 (2), 448-458 (2010).</li>
<li>Jessen, K. R., Mirsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+origin+and+development+of+glial+cells+in+peripheral+nerves%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">The origin and development of glial cells in peripheral nerves.</a> Nat Rev Neurosci. 6 (9), 671-682 (2005).</li>
<li>Mung, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+efficient+generation+of+neural+progenitors+from+adult+bone+marrow+stromal+cells+by+hypoxic+preconditioning%5BTitle%5D)+AND+%22Stem+Cell+Res+Ther%22%5BJournal%5D)">Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning.</a> Stem Cell Res Ther. 7 (1), 146(2016).</li>
<li>Ao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+regeneration+of+transected+sciatic+nerves+of+adult+rats+using+chitosan+nerve+conduits+seeded+with+bone+marrow+stromal+cell-derived+Schwann+cells%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">The regeneration of transected sciatic nerves of adult rats using chitosan nerve conduits seeded with bone marrow stromal cell-derived Schwann cells.</a> Biomaterials. 32 (3), 787-796 (2011).</li>
<li>Tondreau, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+BM+mesenchymal+stem+cells+by+plastic+adhesion+or+negative+selection%3A+phenotype%2C+proliferation+kinetics+and+differentiation+potential%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential.</a> Cytotherapy. 6 (4), 372-379 (2004).</li>
<li>Baksh, D., Song, L., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+mesenchymal+stem+cells%3A+characterization%2C+differentiation%2C+and+application+in+cell+and+gene+therapy%5BTitle%5D)+AND+%22J+Cell+Mol+Med%22%5BJournal%5D)">Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy.</a> J Cell Mol Med. 8 (3), 301-316 (2004).</li>
<li>Jirsova, K., Sodaar, P., Mandys, V., Bar, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cold+jet%3A+a+method+to+obtain+pure+Schwann+cell+cultures+without+the+need+for+cytotoxic%2C+apoptosis-inducing+drug+treatment%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment.</a> J. Neurosci. Methods. 78 (1-2), 133-137 (1997).</li>
</ol>

All authors of this manuscript have no disclosures to declare.

It is essential to preserve the &#34;stemness&#34; of MSCs prior to the enrichment of neural progenitors via hypoxic preconditioning and neurosphere culture. From our experience, multipotent MSCs can be reliably identified by their elongated fibroblast-like morphology. In contrast, MSCs that have adopted a more flattened, quadrangular morphology, with prominent cytoskeletal stress fibers, do not readily adopt neural cell fates and should be discarded. In general, we do not utilize MSCs with passage numbers greater than e...

The transplantation of neural progenitors and their derivatives demonstrates promise as a treatment strategy following traumatic nerve injury1,2 and neurodegeneration3,4. Prior to clinical application, it is essential to ensure: i) a method for accessing and expanding upon an autologous source of stem/progenitor cells and ii) a means to direct them to relevant, mature cell types3. Our interest in cell therapy for spinal cord injury led us to seek a robust, autologous cell source of neural progenitors from adult tissues.
A subpopulation of MSCs originates from the neural crest and is readily accessible from the marrow cavity. These cells are neural progenitors that can generate neurons and glia5. Animal models of cerebral ischemia demonstrate that hypoxia promotes the proliferation and multipotency of neural progenitors within the brain6. This was the basis for utilizing hypoxic preconditioning as a means of expanding upon marrow-derived neural progenitors.
The transplantation of Schwann cells into the injured spinal cord promotes regeneration2. SCLCs can be generated from MSCs by means of supplementation with gliogenic factors (i.e., &#946;-Heregulin, bFGF, and PDGF-AA) but demonstrate phenotypic instability. Upon the withdrawal of growth factors, they revert to a fibroblast-like phenotype7. Phenotypic instability is undesirable in cell transplantation due to the risk of aberrant differentiation and carcinogenesis. As Schwann cell precursors are associated with axon bundles within the embryonic peripheral nerve8, we were led to coculture SCLCs with purified embryonic DRG neurons7,9. Resultant mature Schwann cells are fate-committed and demonstrate function in vitro7,9 and in vivo10.
Our protocol for the enrichment of neural progenitors from MSCs is simple and efficient and results in an increase in cell number for subsequent assays. The derivation of fate-committed Schwann cells via the coculture platform allows for the study of glial differentiation and for the generation of stable and functional Schwann cells for potential clinical application.

<table><tbody><tr><td>&amp;alpha;MEM</td><td>Sigmaaldrich</td><td>M4526</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermofisher scientific</td><td>12400-024</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermofisher scientific</td><td>21103-049</td><td></td></tr><tr><td>FBS</td><td>Biosera</td><td>FB-1280/500</td><td></td></tr><tr><td>B27</td><td>Thermofisher scientific</td><td>17504-001</td><td></td></tr><tr><td>Epidermal growth factor (EGF)</td><td>Thermofisher scientific</td><td>PHG0313</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)&amp;nbsp;</td><td>Peprotech</td><td>100-18B/100UG</td><td></td></tr><tr><td>Nerve growth factor (NGF)&amp;nbsp;</td><td>Millipore</td><td>NC011</td><td></td></tr><tr><td>Platelet-derived growth factor-AA (PDGF-AA)</td><td>Peprotech</td><td>100-13A</td><td></td></tr><tr><td>Heregulin beta-3, EGF domain (&amp;beta;-Her)</td><td>Millipore</td><td>01-201</td><td></td></tr><tr><td>Uridine</td><td>Sigmaaldrich</td><td>U3003</td><td></td></tr><tr><td>5-Fluro-2&#039; - deoxyuridine (FDU)</td><td>Sigmaaldrich</td><td>F0503</td><td></td></tr><tr><td>Poly-D-lysine (PDL)</td><td>Sigmaaldrich</td><td>P7886-1G</td><td></td></tr><tr><td>Laminin</td><td>Thermofisher scientific</td><td>23017015</td><td></td></tr><tr><td>GlutaMAX</td><td>Thermofisher scientific</td><td>35050061</td><td></td></tr><tr><td>Penicillin / streptomycin (P/S)</td><td>Thermofisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>TrypLE Express</td><td>Thermofisher Scientific</td><td>12604-013</td><td></td></tr><tr><td>10 cm plate for adherent culture</td><td>TPP</td><td>93100</td><td>Used for selection of MSCs by tissue culture adherence</td></tr><tr><td>6-well plate for adherent culture</td><td>TPP</td><td>92006</td><td>Used for expansion of MSCs following passaging</td></tr><tr><td>UltraLow 6-well plate for non-adherent culture</td><td>Corning</td><td>3471</td><td>Used for neural progenitor enrichment</td></tr><tr><td>anti-human CD90(Thy-1)</td><td>BD Biosciences</td><td>555593</td><td></td></tr><tr><td>anti-human CD73</td><td>BD Biosciences</td><td>550256</td><td></td></tr><tr><td>anti-human/rat STRO-1</td><td>R&amp;amp;D Systems</td><td>MAB1038</td><td></td></tr><tr><td>anti-human nestin</td><td>R&amp;amp;D Systems</td><td>MAB1259</td><td></td></tr><tr><td>anti-human CD45</td><td>BD Biosciences</td><td>555480</td><td></td></tr><tr><td>anti-rat CD90(Thy-1)</td><td>BD Biosciences</td><td>554895</td><td></td></tr><tr><td>anti-rat CD73</td><td>BD Biosciences</td><td>551123</td><td></td></tr><tr><td>anti-rat nestin</td><td>BD Biosciences</td><td>MAB1259</td><td></td></tr><tr><td>anti-rat CD45</td><td>BD Biosciences</td><td>554875</td><td></td></tr><tr><td>Anti-S100&amp;beta;</td><td>Dako</td><td>Z031101</td><td></td></tr><tr><td>Anti-p75</td><td>Millipore</td><td>MAB5386</td><td></td></tr><tr><td>Anti-GFAP</td><td>Sigmaaldrich</td><td>G3893</td><td></td></tr><tr><td>Anti-Class III-beta tubulin (Tuj-1)</td><td>Covance</td><td>MMS-435P</td><td></td></tr><tr><td>Anti-Human nuclei</td><td>Millipore</td><td>MAB1281</td><td></td></tr><tr><td>Hypoxia chamber</td><td>Billups-Rothenberg</td><td>MIC-101</td><td></td></tr><tr><td>HEPES buffer</td><td>Sigmaaldrich</td><td>H4034-100G</td><td></td></tr></tbody></table>

hypoxic preconditioning of marrow-derived progenitor cells as a source for the generation of mature schwann cells

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

An overview of the key stages in our protocol is illustrated in Figure 1. In summary, rat and human MSCs are selected for by adherence to tissue culture plastic. Expanded MSCs are preconditioned with hypoxia and are then subject to neurosphere-forming conditions. Neurospheres are plated and allowed to differentiate into SCLCs. SCLCs are cocultured with purified DRG neurons to generate fate-committed Schwann cells.
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Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

The overall goal of this cell culture technique is to enrich for bone marrow neural progenitors to facilitate their differentiation into functional Schwann cells. This method addresses a key issue in stem cell and regenerative medicine:how to use a limited source of neuroprogenitors to generate glia to assist in post-traumatic axonal regrowth and remyelination. An advantage of this technique is the use of efficient culture conditioning to facilitate neural cell expansion from bone marrow stromal cells for their use in autologous cell therapy.After two days in culture, remove 75%of the spent medium and the non-adherent cells and rinse the adherent cells with three 10 millimeter PBS washes. After the third wash, replace the PBS with 10 milliliters of marrow stomal cells, or MSC, growth medium and return the culture to the incubator. Visible colonies should appear by day six to seven.On day 10, wash the culture in fresh PBS and detach the cells with 1.5 milliliters of recombinant enzymatic cell dissociation reagent. After five minutes at 37 degrees Celsius, add three milliliters of MSC growth medium to neutralize the reaction and collect the detached cells by centrifugation. Resuspend the pellet in five milliliters of fresh MSC growth medium and seed the cells at a four times 10 to the fourth cells per square centimeter density in a new 10 centimeter tissue culture plate.The cells should reach 80 to 90%confluency within two days of passaging. For hypoxic preconditioning, first release the ring clamp and clean the exposed parts with 70%ethanol. Further disassemble the hypoxia chamber into components and place them into a laminar flow hood for sterilization under UV light for 15 minutes.Next, wash an 80 to 90%confluent MSC culture with 10 milliliters of PBS as just demonstrated and treat the cultures with fresh MSC growth medium supplemented with 25 millimolar hepes. Place the treated culture into the reassembled hypoxia chamber and tighten the ring clamp. Seal the connecting ends of the chamber to ensure that there is no gas leakage and flush a gas mixture of 99%nitrogen and 1%oxygen into the chamber at a flow rate of 10 liters per minute.After five minutes, place the chamber into the cell culture incubator for 16 hours. The next morning, detach the cells as just demonstrated and collect them by centrifugation. Resuspend the pellet in neural progenitor medium and seed six times 10 to the third cells per square centimeter onto low-attachment 6 well plates for their culture at 37 degrees Celsius and 5%CO2 for 12 days.Sizable non-adherent spheres of cells should be observed by day six to seven. With more neurospheres apparent in hypoxia conditioned cultures than those under normoxia by days 10 to 12. On day 12, use a 10 milliliters pipet to transfer the neurospheres into a 15 milliliters conical tube and collect the neurospheres by centrifugation.Resuspend the spheres in DMEM/F12 and plate five to 10 neurospheres per square centimeter onto poly-D-lysine laminin coated 6 well plates in 1.5 milliliters of glial induction medium per well. Maintain the sphere cell culture for at least seven days, refreshing the medium every three days. To harvest the dorsal root ganglia, transfer the first embryo into a 10 centimeter culture dish containing room temperature PBS under a dissecting microscope in the prone position and insert micro dissecting forceps along either side of the spinal cord.Using blunt dissection, separate the spinal cord from the surrounding soft tissue using forceps to excise the spinal cord along the neck opening and tail stub. Remove the residual soft tissue from the freed spinal cord until only the spinal cord, nerve roots, and attached dorsal root ganglion remain. Detach the individual dorsal root ganglia from their connecting nerve roots.Then, use a pipet pen equipped with a one milliliters pipet tip to transfer up to 100 dorsal root ganglia into 1.5 milliliters microcentrifuge tube of PBS. Collect the ganglia by centrifugation and resuspend the pellets in 200 microliters of recombinant enzymatic cell dissociation reagent per tube. After 10 minutes at 37 degrees Celsius, centrifuge the dorsal root ganglia again using a 200 microliter pipet tip to gently triturate the pellets in dorsal root ganglion neuron maintenance medium.Seed the dorsal root ganglion cells at five times 10 to the third cells per square centimeter onto poly-D-lysine laminin coated 6 well plates in 1.5 milliliters of dorsal root ganglion neuron maintenance medium per well. After two days in culture at 37 degrees Celsius and 5%CO2, wash the cells and return the neuronal cell cultures to the incubator in fresh dorsal root ganglion neuron purification medium for another two days. After three to four maintenance and purification cycles, the dorsal root ganglion cell cultures should test positive for the neuronal marker Tuj-1 and negative for the glial cell marker S100 beta.On day seven of the Schwann cell-like cell culture, rinse the cells in PBS followed by dissociation with 0.5 milliliters of recombinant enzymatic cell dissociation reagent per well at 37 degrees Celsius for five minutes. Resuspend the cell pellet in co-culture medium and seed the Schwann cell-like cells onto the purified dorsal root ganglion neuron culture at one times 10 to the third cells per square centimeter for 14 days of co-culture at 37 degrees Celsius and 5%CO2. Healthy MSCs exhibit a tapered morphology in contrast to the quadrangular appearance of MSCs that have been maintained for a high number of passages and have lost their multipotency.Expanded MSC colonies should demonstrate the expression of MSC markers, the absence of hematopoietic stem cell markers, and the capacity for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Healthy MSC culture that have been subjected to hypoxic preconditioning, demonstrate neurospheres that are larger in both number and size. The purity of the dorsal root ganglion neuron network can be confirmed by the expression of the neuronal marker Tuj-1 and the concomitant absence of the Schwann cell marker S100.After 14 days in co-culture with dorsal root ganglion neurons, Schwann cell-like cells acquire the features and functional properties of mature fate-committed Schwann cells. Once mastered, the neural progenitor subpopulation can be expanded in either human or rat BMSCs in 10 days if the cell expansion technique is performed properly. While attempting this procedure, it's important to remember to confirm that the BMSC sample is endowed with nestin-positive neuroprogenitors and the CD45 positive hematopoietic progenitors have all been removed.After watching this video, you should have a good understanding of how to source BMSCs, to enrich for the neural progenitor subpopulation, and to direct neural progenitor differentiation into fate-committed Schwann cells for the use in enhancing post-traumatic axonal regrowth and remyelination. Following this procedure, myelinating glia, such as mature Schwann cells, can be derived for their use in the development of therapies for demyelinating injuries and diseases of both the peripheral and central nervous system.

hypoxic-preconditioning-marrow-derived-progenitor-cells-as-source-for

Research

JoVE Journal

Developmental Biology

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

This video demonstrates a method for obtaining Schwann cell-like cells from multipotent bone marrow stromal cells (BMSCs). Initially, the BMSCs undergo hypoxia treatment, followed by directed differentiation into neuronal progenitor cells. Subsequently, specific media and culture conditions are employed to induce the transformation of these progenitor cells into Schwann cell-like cells.

Generating Schwann Cell-Like Cells from Bone Marrow Stromal Cells

This video demonstrates a procedure for isolating dorsal root ganglion (DRG) neurons from rat embryos and co-culturing them with Schwann cell precursors to generate mature Schwann cells. The isolated DRGs are processed to isolate neurons and glial cells. The isolated cells are cultured to purify the neurons, which are then co-cultured with Schwann cell-like cells (SCLCs) in a co-culture medium. Interaction with the DRG neurons leads to the maturation of SCLCs into Schwann cells.

Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann 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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Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer’s disease.

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

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.

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Learn hypoxic preconditioning of marrow-derived progenitor cells to generate mature Schwann cells for regenerative medicine. This method enables autologous cell therapy for nerve injury repair and remyelination.

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

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