Name: hypoxic preconditioning of marrow-derived progenitor cells as a source for the generation of mature schwann cells
Uploaded: 2017-06-14T13:00:00.000+00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells at JoVE.com

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>

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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 eight. To preserve their stemness, it is critical to promptly passage MSCs before they reach 100% confluence. Conversely, maintaining MSCs at a too-low confluence is undesirable. From our experience, seeding MSCs at a density of 40,000 cells/cm2, or simply passaging cells that are 80% confluent at a 1:2 ratio, allows for the best results.
The proper establishment and maintenance of the DRG network is a critical determinant of coculture success. The time required for DRG harvest should be kept to a minimum. Individual ganglia should be handled in an atraumatic manner, particularly during detachment from the spinal cord, when it is best to handle the nerve roots only. In general, we aim for a period of less than 2 h between the time of animal sacrifice and the enzymatic digestion of harvested DRGs, as a prolonged harvest results in tissue maceration and the loss of cell viability. The detachment of DRG neurons from the substratum during culture is often encountered. To prevent this from occurring, the coating should be freshly prepared and performed near the time of tissue harvest. In general, large, undigested DRG clusters detach more often and do not yield coculture success. The duration of enzymatic digestion and the amount of trituration can be adjusted, with the aim of achieving a network with an appearance resembling Figure 5.
While our coculture platform consistently induces fate commitment, only 20-30% of cultures performed in parallel yield fate-committed Schwann cells7,13. We therefore prepare enough DRGs and SCLCs to concomitantly perform cocultures in three to four 6-well culture plates. We hypothesize that a combination of factors related to the underlying DRG network, including their embryonic age, cell viability, density, and topography, affect coculture success. These underlying variables need to be further investigated and standardized. Apart from limitations in the coculture yield, a means of superseding the requirement for rat-derived DRG neurons and animal products should be sought. Furthermore, the duration of the protocol should be shortened. As an abbreviated modification of our protocol, we have had success in deriving mature Schwann cells from day 10 neurospheres seeded directly onto purified DRG neurons, without the prior generation of SCLCs7,10.
Neurospheres enriched via our method serve as a robust source of cells of neuronal and glial lineages. Our platform for directing precursor cells to fate commitment has the advantage of avoiding genetic manipulation, with its inherent risks, and the resultant cells are of relevance for cell transplantation, disease modeling, and the study of glial differentiation.

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

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.
The morphology of cultured rat and human MSCs is illustrated in Figure 2. Their healthy tapered morphology is shown in contrast to the quadrangular appearance of MSCs maintained for high passage numbers, which have lost their multipotency. Expanded rat and human colonies should demonstrate the expression of MSC markers, an absence of hematopoietic stem cell markers, and the capacity for trilineage differentiation (Figure 3). Healthy MSCs from between passages 3 and 8 are subject to hypoxic preconditioning for 16 h and are thereafter passaged onto low-adherence culture plates with EGF/bFGF supplementation. Hypoxic preconditioning results in larger numbers of neurospheres, as well as larger average neurosphere sizes (Figure 4).
Neurospheres are plated onto PDL/laminin-coated culture plates and induced to become SCLCs by culture in glial induction medium containing &#946;-Heregulin, bFGF, and PDGF-AA. SCLCs exhibit the characteristic tapered morphology of Schwann cells and the corresponding marker expression, yet they are phenotypically unstable and revert to a fibroblast phenotype upon the discontinuation of growth factors
Coculture with sensory neurons is a prerequisite to bring about a cell-intrinsic switch to fate commitment. The establishment of purified DRG networks is achieved by means of pulsed treatment with FDU and uridine to remove endogenous glia and should be confirmed by the absence of S100&#946; immunopositivity (Figure 5). On day 7, SCLCs are passaged and cocultured with purified DRG neurons for 14 days (Figure 6). Upon the completion of coculture, mature, fate-committed Schwann cells should emerge.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55794/55794fig1.jpg" /> 
Figure 1: Overview of the protocol. Bone marrow is obtained from either rat femurs or human iliac crest aspirates. MSCs from within the bone marrow can attach and expand upon tissue culture plastic. In order to enrich for MSCs with neural potential, cells are preconditioned in 1% O2 for 16 h and are then passaged onto low-adherence culture plates with bFGF/EGF supplementation. This results in the formation of neurospheres, which are plated onto PDL/laminin-coated tissue culture plastic and cultured in glial induction medium to generate SCLCs. SCLCs are passaged and cocultured with purified DRG neurons for 2 weeks in order to direct them to maturity. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55794/55794fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55794/55794fig2.jpg" /> 
Figure 2: Establishment of MSC colonies. Sizeable MSC colonies should be visible 6-7 days after the plating of bone marrow cells onto tissue culture plastic. A representative image of a rat MSC colony is shown (A), while human colonies demonstrate a similar appearance. Colonies can be passaged on day 10. Seen at higher magnification, both rat (B) and human (D) MSCs exhibit a characteristic fibroblast-like morphology after passaging. MSCs that are maintained for high passage numbers acquire a flattened, quadrangular morphology (C) and should be discarded. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55794/55794fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55794/55794fig3.jpg" /> 
Figure 3: Characterization of MSCs. (A-D) Representative images of human MSCs. MSCs can be characterized by the expression of appropriate markers, such as CD90 (A), CD73 (B), and Stro-1 (C), and by the absence of hematopoietic stem cell markers, such as CD45 (D). (E-G) Rat MSCs isolated and expanded as described in the protocol demonstrate multipotency in their ability to form adipocytes (E; fat deposits stained with Sudan Red), osteoblasts (F; pericellular matrix stained with Alizarin Red), and chondrocytes (G; proteoglycans stained with Safranin-O) under appropriate culture conditions. Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55794/55794fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55794/55794fig4.jpg" /> 
Figure 4: Enrichment of neural progenitors from MSCs. Both rat (A) and human (C) MSCs form neurospheres when cultured upon low-adherence tissue culture plastic in medium supplemented with EGF/bFGF. The numbers and average diameter of rat (B) and human (D) spheres is enhanced via the hypoxic preconditioning of MSCs (16 h, 1% O2) prior to sphere induction. Scale bars = 200 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55794/55794fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55794/55794fig5.jpg" /> 
Figure 5: Establishing purified rat DRG networks. Purified DRG networks are established after pulsed treatment with the antimitotic agents FDU and uridine (A). Neurite networks that are devoid of S100&#946;-expressing endogenous glia (B) are ready for coculture with SCLCs. Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55794/55794fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55794/55794fig6.jpg" /> 
Figure 6: Generation of bone marrow-derived Schwann cells via coculture with DRG neurons. Upon completing 2 weeks of coculture with purified DRG neurons and human SCLCs, spindle-shaped, fate-committed Schwann cells emerge from both normoxia- and hypoxia-treated groups (A and D). These cells express the Schwann cell markers p75 (B and E) and S100&#946; (C and F). The expression of human nuclei antigen (HuNeu) demonstrates that the S100&#946;-positive cells were not contaminating glial cells originating from rat DRGs. Scale bars = 100 &#181;m.

Watch this Scientific Journal Video about Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells at JoVE.com

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

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.3K Views.  The University of Hong Kong. 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. 

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

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Neuroscience

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Immunology and Infection

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

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

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

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.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Biology

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Bioengineering

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

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.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

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

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

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.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

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

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

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&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Utilizing Low Oxygen Tension to Reduce Hematopoietic Cells in Murine Bone Marrow Stromal Cell Cultures

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this reason, BMSCs, which support hematopoiesis and contribute to all the functions of the skeleton, continue to be widely used to study multipotent mesenchymal progenitors (MMPs) and to infer SSC function. Moreover, given the breadth of transgenic murine models used to study musculoskeletal diseases, the use of BMSCs also serves as a powerful tool to examine the molecular mechanisms regulating MMPs and SSCs. However, common isolation procedures for murine BMSCs result in over 50% of recovered cells being of hematopoietic origins, potentially hindering the interpretation of the data generated during these studies. Here, we describe a method using low oxygen tension or hypoxia for the selective elimination of CD45+ cells in BMSC cultures. Importantly, this method can be easily implemented to not only reduce hemopoietic contaminants but to also enhance the percentage of MMPs and putative SSCs in BMSC cultures.

A routine culture of bone marrow stromal cells (BMSCs) leads to the isolation of heterogeneous cell populations, with many cells being of hematopoietic origins. Here, we describe a method that utilizes low oxygen tension to greatly reduce hematopoietic contaminants in murine BMSC cultures.

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this ...

Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells

Source: Tsui, Y, et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55794">Hypoxic Preconditioning of Marrow-derived Progenitor Cells as a Source for the Generation of Mature Schwann Cells.</a> J. Vis. Exp. (2017).
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.

Izolacja neuronów DRG i kokultura z prekursorami komórek Schwanna w celu wytworzenia komórek Schwanna

Generation of Fate-committed Schwann Cells via Coculture with DRG Neurons
1. Preparation of purified rat DRG neurons

	Autoclave all dissection tools ...