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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+transplantation:+a+repair+strategy+for+spinal+cord+injury?.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+transplantation+for+spinal+cord+injury+repair:+its+significant+therapeutic+potential+and+prospectus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+human+neurodegenerative+disorders--time+for+clinical+translation?.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+Differentiation+of+Oligodendrocyte+Progenitor+Cells+From+Mouse+Induced+Pluripotent+Stem+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroepithelial+cells+supply+an+initial+transient+wave+of+MSC+differentiation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem/progenitor+cells+participate+in+the+regenerative+response+to+perinatal+hypoxia/ischemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+Schwann+cells+achieve+fate+commitment--a+prerequisite+for+remyelination+therapy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+development+of+glial+cells+in+peripheral+nerves.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+generation+of+neural+progenitors+from+adult+bone+marrow+stromal+cells+by+hypoxic+preconditioning.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regeneration+of+transected+sciatic+nerves+of+adult+rats+using+chitosan+nerve+conduits+seeded+with+bone+marrow+stromal+cell-derived+Schwann+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+BM+mesenchymal+stem+cells+by+plastic+adhesion+or+negative+selection:+phenotype,+proliferation+kinetics+and+differentiation+potential.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mesenchymal+stem+cells:+characterization,+differentiation,+and+application+in+cell+and+gene+therapy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+jet:+a+method+to+obtain+pure+Schwann+cell+cultures+without+the+need+for+cytotoxic,+apoptosis-inducing+drug+treatment.">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 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 border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">&#945;MEM</td>
			<td style="width:140px;">Sigmaaldrich</td>
			<td style="width:101px;">M4526</td>
			<td style="width:329px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12</td>
			<td>Thermofisher scientific</td>
			<td>12400-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal medium</td>
			<td>Thermofisher scientific</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Biosera</td>
			<td>FB-1280/500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27</td>
			<td>Thermofisher scientific</td>
			<td>17504-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal growth factor (EGF)</td>
			<td>Thermofisher scientific</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic fibroblast growth factor (bFGF)&#160;</td>
			<td>Peprotech</td>
			<td>100-18B/100UG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nerve growth factor (NGF)&#160;</td>
			<td>Millipore</td>
			<td>NC011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet-derived growth factor-AA (PDGF-AA)</td>
			<td>Peprotech</td>
			<td>100-13A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heregulin beta-3, EGF domain (&#946;-Her)</td>
			<td>Millipore</td>
			<td>01-201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Uridine</td>
			<td>Sigmaaldrich</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Fluro-2&#39; - deoxyuridine (FDU)</td>
			<td>Sigmaaldrich</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-D-lysine (PDL)</td>
			<td>Sigmaaldrich</td>
			<td>P7886-1G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin</td>
			<td>Thermofisher scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMAX</td>
			<td>Thermofisher scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin / streptomycin (P/S)</td>
			<td>Thermofisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE Express</td>
			<td>Thermofisher Scientific</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">6-well plate for adherent culture</td>
			<td>TPP</td>
			<td>92006</td>
			<td>Used for expansion of MSCs following passaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraLow 6-well plate for non-adherent culture</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Used for neural progenitor enrichment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human CD90(Thy-1)</td>
			<td>BD Biosciences</td>
			<td>555593</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human CD73</td>
			<td>BD Biosciences</td>
			<td>550256</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human/rat STRO-1</td>
			<td>R&#38;D Systems</td>
			<td>MAB1038</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human nestin</td>
			<td>R&#38;D Systems</td>
			<td>MAB1259</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>555480</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD90(Thy-1)</td>
			<td>BD Biosciences</td>
			<td>554895</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD73</td>
			<td>BD Biosciences</td>
			<td>551123</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat nestin</td>
			<td>BD Biosciences</td>
			<td>MAB1259</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD45</td>
			<td>BD Biosciences</td>
			<td>554875</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-S100&#946;</td>
			<td>Dako</td>
			<td>Z031101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-p75</td>
			<td>Millipore</td>
			<td>MAB5386</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-GFAP</td>
			<td>Sigmaaldrich</td>
			<td>G3893</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Class III-beta tubulin (Tuj-1)</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Human nuclei</td>
			<td>Millipore</td>
			<td>MAB1281</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hypoxia chamber</td>
			<td>Billups-Rothenberg</td>
			<td>MIC-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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

7.2K 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 Cardiac Progenitor Cells from Embryonic Stem Cells

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

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

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

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

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

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

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

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

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

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

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

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

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

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

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

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

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

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

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

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

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

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

Summary

Explore More Videos

Chapters in this video

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

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

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Single-cell Photoconversion in Living Intact Zebrafish

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

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

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage