This work was supported by the Alexander von Humboldt Foundation Sofja Kovalevskaja Award to VLK.

<ol>
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S., Clouston, A. D., Theise, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductular+reactions+in+human+liver:+diversity+at+the+interface.">Ductular reactions in human liver: diversity at the interface.</a> Hepatology. 54 (5), 1853-1863 (2011).</li><li>Tarlow, B. D., Finegold, M. J., Grompe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+tracing+of+Sox9++liver+progenitors+in+mouse+oval+cell+injury.">Clonal tracing of Sox9+ liver progenitors in mouse oval cell injury.</a> Hepatology. 60 (1), 278-289 (2014).</li><li>Shin, S., 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=Foxl1-Cre-marked+adult+hepatic+progenitors+have+clonogenic+and+bilineage+differentiation+potential.">Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential.</a> Genes Dev. 25 (11), 1185-1192 (2011).</li><li>Furuyama, K., 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=Continuous+cell+supply+from+a+Sox9-expressing+progenitor+zone+in+adult+liver,+exocrine+pancreas+and+intestine.">Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine.</a> Nat Genet. 43 (1), 34-41 (2011).</li><li>Font-Burgada, 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=Hybrid+Periportal+Hepatocytes+Regenerate+the+Injured+Liver+without+Giving+Rise+to+Cancer.">Hybrid Periportal Hepatocytes Regenerate the Injured Liver without Giving Rise to Cancer.</a> Cell. 162 (4), 766-779 (2015).</li><li>Kuramitsu, K., 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=Failure+of+fibrotic+liver+regeneration+in+mice+is+linked+to+a+severe+fibrogenic+response+driven+by+hepatic+progenitor+cell+activation.">Failure of fibrotic liver regeneration in mice is linked to a severe fibrogenic response driven by hepatic progenitor cell activation.</a> Am J Pathol. 183 (1), 182-194 (2013).</li><li>Pritchard, M. T., Nagy, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+fibrosis+is+enhanced+and+accompanied+by+robust+oval+cell+activation+after+chronic+carbon+tetrachloride+administration+to+Egr-1-deficient+mice.">Hepatic fibrosis is enhanced and accompanied by robust oval cell activation after chronic carbon tetrachloride administration to Egr-1-deficient mice.</a> Am J Pathol. 176 (6), 2743-2752 (2010).</li><li>Spee, B., 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=Characterisation+of+the+liver+progenitor+cell+niche+in+liver+diseases:+potential+involvement+of+Wnt+and+Notch+signalling.">Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling.</a> Gut. 59 (2), 247-257 (2010).</li><li>Eckert, C., 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=Podoplanin+discriminates+distinct+stromal+cell+populations+and+a+novel+progenitor+subset+in+the+liver.">Podoplanin discriminates distinct stromal cell populations and a novel progenitor subset in the liver.</a> Am J Physiol Gastrointest Liver Physiol. 310 (1), G1-G12 (2016).</li><li>Epting, C. 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=Stem+cell+antigen-1+is+necessary+for+cell-cycle+withdrawal+and+myoblast+differentiation+in+C2C12+cells.">Stem cell antigen-1 is necessary for cell-cycle withdrawal and myoblast differentiation in C2C12 cells.</a> J Cell Sci. 117 (Pt 25), 6185-6195 (2004).</li><li>Tirnitz-Parker, J. E., Tonkin, J. N., Knight, B., Olynyk, J. K., Yeoh, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+immortalisation+of+hepatic+oval+cells+from+adult+mice+fed+a+choline-deficient,+ethionine-supplemented+diet.">Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet.</a> Int J Biochem Cell Biol. 39 (12), 2226-2239 (2007).</li><li>Rountree, C. B., 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=A+CD133-expressing+murine+liver+oval+cell+population+with+bilineage+potential.">A CD133-expressing murine liver oval cell population with bilineage potential.</a> Stem Cells. 25 (10), 2419-2429 (2007).</li><li>Rountree, C. B., Ding, W., Dang, H., Vankirk, C., Crooks, G. M. <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+CD133++liver+stem+cells+for+clonal+expansion.">Isolation of CD133+ liver stem cells for clonal expansion.</a> J Vis Exp. (56), (2011).</li><li>Sidney, L. E., McIntosh, O. D., Hopkinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+Change+and+Induction+of+Cytokeratin+Expression+During+In+Vitro+Culture+of+Corneal+Stromal+Cells.">Phenotypic Change and Induction of Cytokeratin Expression During In Vitro Culture of Corneal Stromal Cells.</a> Invest Ophthalmol Vis Sci. 56 (12), 7225-7235 (2015).</li><li>Hass, R., Kasper, C., Bohm, S., Jacobs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+populations+and+sources+of+human+mesenchymal+stem+cells+(MSC):+A+comparison+of+adult+and+neonatal+tissue-derived+MSC.">Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC.</a> Cell Commun Signal. 9, 12 (2011).</li><li>Schmelzer, E., 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=Human+hepatic+stem+cells+from+fetal+and+postnatal+donors.">Human hepatic stem cells from fetal and postnatal donors.</a> J Exp Med. 204 (8), 1973-1987 (2007).</li></ol>

The authors have no competing financial interests.

Liver inflammation and injury of different origins trigger regenerative processes in the liver that are accompanied by progenitor cell expansion and activation2,3. These liver progenitor cells possess stem cell characteristics and likely play a significant role in the pathomechanism of various liver diseases.
The heterogeneity of liver progenitor cells has long been suggested. The re-evaluation of liver progenitor subsets using a novel...

Liver regeneration is mostly associated with the self-renewal capacity of hepatocytes. Nevertheless, chronic liver injuries occur with progenitor cell activation and expansion, which have been associated with their ability to differentiate into hepatocytes and cholangiocytes1,2,3,4. This is especially relevant because, during chronic injuries, hepatocyte proliferation is not effective. Despite multiple genetic tracing studies targeting progenitor cells, their role in liver regeneration remains controversial5,6,7,8. Moreover, the activation of progenitor cells has been linked to increased fibrotic response in the liver, which raises questions about their exact role during injuries9,10.
The heterogeneous nature of the progenitor cell compartment has long been suggested by gene expression studies that isolated progenitor cells expressing a single surface marker using microdissection or cell sorting-based methods1,11. Indeed, recently, a novel surface marker combination using gp38 (podoplanin) unequivocally linked previous single markers of progenitor cells to various subsets12. Importantly, these subsets not only differed in their surface marker expression but also exhibited functional alterations during injuries12.
Multiple animal models have been utilized to investigate progenitor cell activation and liver regeneration. It seems that the various injury types promote the activation of differing subsets of progenitor cells12. This might explain the phenotypic divergence of the ductular reaction observed in humans4. Thus, the complex phenotypic and functional analyses of progenitor cells are pivotal to understand their role in injuries and the true significance of the ductular reaction in liver diseases.
Besides surface marker combinations, the crucial differences in cell isolation protocols further complicate the conclusions based on previous studies2. A substantial amount of studies addressed the role of progenitor cells that greatly differ in their isolation protocol (e.g., liver dissociation (enzyme combination and duration of the process), density medium, and centrifugation speed)2. An optimized isolation technique, providing better viability for rare cell populations and reflective of subset composition, has been developed and published recently12. The aim of this article is to provide a more detailed protocol of this liver cell isolation procedure and the subset analysis to allow for the proper reproduction of the technique. Additionally, the protocol includes a comparison with the previous isolation method to demonstrate the differences compared to the new protocol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI</td>
			<td>Life Technologies</td>
			<td>21875-034</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol red free DMEM</td>
			<td>Life Technologies</td>
			<td>31053-028</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase P</td>
			<td>Sigma-Aldrich</td>
			<td>11249002001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse-I</td>
			<td>Sigma-Aldrich</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Life Technologies</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Life Technologies</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14025-050</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>Prepare 1 % stock solution</td>
		</tr>
		<tr>
			<td>10 % BSA</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>autoMACS Rinsing Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>add 0.5 % (v/v) BSA and store on ice</td>
		</tr>
		<tr>
			<td>Phenol-red free DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D1145</td>
			<td></td>
		</tr>
		<tr>
			<td>counting Beads Count Bright</td>
			<td>Life Technologies</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr>
			<td>PI</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-233</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR Blocking Reagent</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD31 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-418</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Dead Cell Removal Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-485</td>
			<td></td>
		</tr>
		<tr>
			<td>CD64 Purified</td>
			<td>BioLegend</td>
			<td>139302</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>CD16/32 Purified</td>
			<td>BioLegend</td>
			<td>101302</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>CD45 APC/Cy7</td>
			<td>BioLegend</td>
			<td>103116</td>
			<td>Dilution: 1:200, marks hematopoetic cells</td>
		</tr>
		<tr>
			<td>CD31 Biotin</td>
			<td>BioLegend</td>
			<td>102504</td>
			<td>Dilution: 1:200, marks endothelial cells</td>
		</tr>
		<tr>
			<td>ASGPR1 Purified</td>
			<td>Bio-Techne</td>
			<td>AF2755-SP</td>
			<td>Dilution: 1:100, marks hepatocytes</td>
		</tr>
		<tr>
			<td>Podoplanin APC</td>
			<td>BioLegend</td>
			<td>127410</td>
			<td>Dilution: 1:1400, marks progenior cells</td>
		</tr>
		<tr>
			<td>Podoplanin Biotin</td>
			<td>BioLegend</td>
			<td>127404</td>
			<td>Dilution: 1:1400</td>
		</tr>
		<tr>
			<td>CD133 PE</td>
			<td>Miltenyi Biotec</td>
			<td>130-102-210</td>
			<td>use 3 &#181;l, marks progenitor cells</td>
		</tr>
		<tr>
			<td>CD133 Biotin</td>
			<td>BioLegend</td>
			<td>141206</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>CD34 Biotin</td>
			<td>eBioScience</td>
			<td>13-0341-81</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>CD90.2 Pacific Blue</td>
			<td>BioLegend</td>
			<td>140306</td>
			<td>Dilution: 1:800</td>
		</tr>
		<tr>
			<td>CD157 PE</td>
			<td>BioLegend</td>
			<td>140203</td>
			<td>Dilution: 1:600</td>
		</tr>
		<tr>
			<td>EpCAM Brilliant Violet 421</td>
			<td>BioLegend</td>
			<td>118225</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Sca-1 Biotin</td>
			<td>Miltenyi Biotec</td>
			<td>130-101-885</td>
			<td>use 10 &#181;l</td>
		</tr>
		<tr>
			<td>Mouse IgG2b, &#954; PE</td>
			<td>BioLegend</td>
			<td>400311</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG1 PE</td>
			<td>BioLegend</td>
			<td>400407</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2b, &#954; APC/Cy7</td>
			<td>BioLegend</td>
			<td>400624</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a, &#954; Biotin</td>
			<td>BioLegend</td>
			<td>400504</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a, &#954; Brilliant Violet 421</td>
			<td>BioLegend</td>
			<td>400535</td>
			<td></td>
		</tr>
		<tr>
			<td>Syrian Hamster IgG APC</td>
			<td>BioLegend</td>
			<td>402012</td>
			<td></td>
		</tr>
		<tr>
			<td>Syrian Hamster IgG Biotin</td>
			<td>BioLegend</td>
			<td>402004</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat IgG Control Purified</td>
			<td>Bio-Techne</td>
			<td>AB-108-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A11055</td>
			<td>Dilution: 1:800</td>
		</tr>
		<tr>
			<td>Streptavidin Alexa Fluor 405</td>
			<td>Life Technologies</td>
			<td>S32351</td>
			<td>Dilution: 1:400</td>
		</tr>
		<tr>
			<td>100 &#181;m Filter mesh</td>
			<td>A. Hartenstein</td>
			<td>PAS3</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSQuant Analyzer 10</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-343</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Pro Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-545</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS AriaTMIII</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACSDiva sofware</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Round bottom tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy plus mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td>RLT lysis buffer is included</td>
		</tr>
	</tbody>
</table>

isolation and enrichment of liver progenitor subsets identified by a novel surface marker combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

The procedure presented here for the digestion of the liver using a novel mixture of enzymes results in a single-cell suspension containing parenchymal and non-parenchymal liver cells (Figures 1 and 2a). After the ACK-lysing of red blood cells, the direct flow cytometry analysis of the single-cell suspension is possible (Figures 1 and 2). The gating strategy involves the exclusion of doublets and dead cells (Figur...

Watch this Scientific Journal Video about Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination at JoVE.com

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

isolation-enrichment-liver-progenitor-subsets-identified-novel

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JoVE Journal

Developmental Biology

10.9K Views.  Saarland University Medical Center. Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

Video: Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

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

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

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

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

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

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

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

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

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...