The authors wish to thank Alison Logar for her excellent technical assistance with flow cytometry. This work was supported by grants from the Department of Defense (J.H.), the Henry J. Mankin Endowed Chair (J.H.), and the Ministry of Education and Science of the Republic of Kazakhstan (A.S.). C.W.C. was supported in part by the American Heart Association predoctoral fellowship (11PRE7490001). M.Corselli was supported by the California Institute for Regenerative Medicine training grant (TG2-01169).

1. Muscle Biopsy Processing
<ol>
	<li>Preserve human skeletal muscle biopsy on ice in Dulbecco&#39;s Modified Eagle Medium (DMEM)supplemented with 5% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (P/S) during transportation.</li>
	<li>After the receipt of the muscle biopsy, remove the specimen from the transportation container and wash it twice in phosphate-buffered saline (PBS) supplemented with 2% antibiotic-antifungal solution (A/A) under sterile conditions.</li>
	<li>Remove the attached adipose and connective tissue with sterile scissors and forceps in DMEM supplemented with 2% A/A.</li>
	<li>Remove large blood vessels under dissection microscope and subsequently cut the muscle specimen into small pieces (&#60; 1 cm2 in size).</li>
	<li>Preserve cut muscle pieces (&#60;5 grams) in 20 ml preservation medium (PM; DMEM supplemented with 10% FBS and 1% P/S) at 4 &#8451; for up to 5 days.</li>
</ol>
2. Muscle Dissociation and Cell Isolation
<ol>
	<li>On the day of cell isolation, remove muscle pieces from PM and wash twice in PBS supplemented with 2% P/S. To make the digestion solution, add type-I, type-II, and type-IV collagenases (100 mg/ml) freshly into PM.</li>
	<li>Finely chop and mechanically mince muscle pieces with sterile scissors and forceps in a petri-dish with a small amount of PM until the solution passes 10 ml serological pipette with no clotting. Remove the residual adipose and connective tissue during this process. Use at least 8-10 grams of adult muscle or 2 grams of fetal muscle for each cell isolation.</li>
	<li>Transfer 4-5 grams of minced adult muscle to 20 ml (or 2 grams of minced fetal muscle into 10 ml) ofthe digestion solution with a 10 ml serological pipette. Digest for 50 - 60 min at 37 &#8451; on an orbital shaker at 70 - 80 rpm. Optimize cell yield and surface antigen preservation by adjusting the digestion time and/or agitation speed accordingly based on the amount of tissue. Observe the digestion status every 15-20 min until the turbidity almost clears.</li>
	<li>Vigorously pipette the digested tissue 3-5 times with a 10 ml serological pipette. Add equal amount of PM to stop the reaction and then centrifuge at 400 x g for 4 min.</li>
	<li>Carefully remove the supernatant; resuspend the pellet with 10 ml PM to wash, and then filtered through a 100&#160;&#956;m cell strainer.</li>
	<li>Centrifuge at 400 x g for 4 min and carefully remove the supernatant. Resuspend pellets in erythrocyte lysis buffer (155 mM NH4Cl, 10mM KHCO3, 0,1mM EDTA), filter through a 70&#160;&#956;m cell strainer to obtain a single cell suspension, and then incubate for 10 min at RT. Filter through a 70-&#61549;m cell strainer again if any precipitation is observed.</li>
	<li>Centrifuge at 400 x g for 4 min and resuspend the cell pellet in 0.5 ml PBS. Count the number of cells. Obtain a total of at least 5 million cells for cell sorting. Dilute the single cell suspension to less than 5 million cells per ml with PBS for staining. Take out 50-100 &#956;l of the cell suspension and split into 11 tubes for control stainings (unstained control, negative controls, and single-color positive controls).</li>
</ol>
3. Cell Labeling and Sorting
<ol>
	<li>Incubate the single cell suspension for 10 min at 4 &#8451; in mouse serum (diluted 1:10 in PBS) for blocking purpose if necessary.</li>
	<li>Add CD34-APC,CD45-APC-Cy7, CD56-PE-Cy7, CD144-PE, and CD146-FITC (all 1:100) into the single cell suspension and incubate for 20 min at 4 &#8451;. For negative control, add equivalent concentrations of APC-, APC-Cy7-, PE-Cy7-, PE-, and FITC-conjugated isotype IgG antibodies and incubate for 20 min at 4 &#8451;. For single-color positive controls, add equivalent concentrations of CD34-APC, CD45-APC-Cy7, CD56-PE-Cy7, CD144-PE, and CD146-FITC individually into each tube and incubate for 20 min at 4 &#8451;.</li>
	<li>After incubation, centrifuge at 400 x g for 4 min to wash. Resuspend the cell pellet in 1 - 2 ml DMEM supplemented with 5% FBS and 1% P/S. The final concentration of the cell suspension should be less than 5 million cells per ml. Add 7-AAD (1:100) and incubate for 15 min at RT for dead cell exclusion. For negative control and single-color positive controls, resuspend cell pellets in 0.5 ml DMEM supplemented with 5% FBS and 1% P/S.</li>
	<li>Transfer all cell suspensions to round-bottom polystyrene flow cytometry tubes. Prepare cell collection tubes (each tube is pre-filled with 500 &#956;l of the appropriate culture medium: MPM for MECs; EGM-2 for PCs; AC medium for ACs). Transport cell suspensions on ice to the cell sorter.</li>
	<li>Run cell suspensions on the cell sorter in the order of the unstained control, negative controls, single-color positive controls, and the main cell suspension while adjusting laser intensity, channel compensation, and cell population gating stringently to maximize cell purity (please refer to articles published by JoVE and other journals for details of flow cytometry).</li>
	<li>Collect desired cell populations in the appropriate collection tubes. Store collected cells at&#160;4 &#8451; if not seeding immediately.</li>
</ol>
4. Post-sorting Cell Culture
<ol>
	<li>Seed freshly sorted MECs (P0) at &#60;10,000 cells/cm2in MPM onto plates pre-coated with type-I collagen. For subsequent passaging of MECs, seed 3,500 - 4,000 cells/cm2 in MPM onto plates/flasks pre-coated with type-I collagen.</li>
	<li>Seed freshly sorted PCs (P0) at &#60;20,000 cells/cm2 in EGM-2 onto plates freshly coated with 0.2% gelatin. For subsequent passaging of PCs, split cells at 1:3 ratio in PC medium (DMEM supplemented with 20% FBS and 1% P/S) onto regular polystyrene culture plates until P2. From P3 onward, seed cells at 6,500 - 7,000 cells/cm2 in PC medium onto regular polystyrene culture plates/flasks.</li>
	<li>Seed freshly sorted ACs (P0) at &#60;20,000 cells/cm2 in AC medium (DMEM supplemented with 20% FBS and 1% P/S) onto regular polystyrene culture plates. For subsequent passaging of ACs, seed cells at 6,500 - 7,000 cells/cm2 in AC medium onto regular polystyrene culture plates/flasks.</li>
</ol>

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J.H. received remuneration from Cook MyoSite, Inc. for consulting services and for royalties received from technology licensing during the period that the above research was performed. All other authors have no conflict of interest to disclose.

Identification and purification of hBVSC subpopulations represent a major advance in the comprehension of MSC ontogeny. There is increasing evidence indicating the perivascular origin of MSCs and the association between tissue-specific precursor cells and blood vessels22-25.&#160;In addition, the capacity to isolate homogeneous subpopulations of hBVSCs further aids the understanding of MSC heterogeneity and vascular cell biology26.
In the past few years, MECs, PCs, and AC...

Human skeletal muscle has been considered a clinically attractive source of stem/progenitor cells. Skeletal muscle contains not only committed myogenic progenitors, skeletal myoblasts, but also primitive myogenic stem cells, including satellite cells and muscle-derived stem cells (MDSCs)1.&#160;The use of human muscle-derived stem/progenitor cells, autologous or allogeneic, in regenerative medicine has been extensively investigated in pre-clinical animal models and clinical trials. The regenerative applications of muscle stem/progenitor cells range from regenerating the dystrophic muscle in Duchenne muscular dystrophy (DMD) patients to repairing the injured heart in patients with heart attack.
Since the discovery of mesenchymal stem/stromal cells (MSCs) and other multipotent precursor cell populations, including bone marrow-derived multipotent adult progenitor cells(MAPCs) and adipose-derived stem cells (ADSCs), adult stem/progenitor cells have been extensively investigated to date1-9.&#160;Nevertheless, their native identity and localization in situ have been obscured by the retrospective isolation methods. Recently, using fluorescence-activated cell sorting (FACS), we and other groups have prospectively identified and purified three multipotent precursor cell populations from blood vessels within human skeletal muscle and several other organs: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs)10.&#160;These three subpopulations of human blood-vessel-derived stem cells (hBVSCs) can be respectively found in the three structural layers of blood vessels: tunica intima, tunica media, and tunica adventitia. More specifically, MECs and PCs are detected in microvessels and capillaries while ACs are localized in the adventitia layer of larger arteries and veins. Each precursor cell subset expresses a unique combination of cell surface antigens: MECs (CD34+/56+/144+/45-), PCs (CD146+/34-/45-/56-), and ACs (CD34+/31-/45-/56-/146-).
Further characterization of these hBVSC subsets revealed that all three precursor cell populations possess mesodermal developmental potentials similar to typical MSCs, including skeletal myogenesis, osteogenesis, chondrogenesis, and adipogenesis. All hBVSC subsets also exhibit classic MSC markers, including CD44, CD73, CD90, and CD105, freshly and in culture. Collectively these pieces of evidence supported the vascular origin of MSCs. Moreover, the therapeutic capacities of MECs, PCs, and ACs have recently been demonstrated in separate studies. MECs sorted from adult human muscle biopsies were shown to regenerate injured and dystrophic skeletal muscles and repair injured myocardium more efficiently than skeletal myoblasts and vascular endothelial cells (ECs). Purified PCs from different human organs have also been shown to repair/regenerate injured and dystrophic skeletal muscles and contribute to the satellite cell pool13-16. Very recently, we have demonstrated that PCs derived from human skeletal muscle effectively repair the infarcted myocardium through indirect paracrine effect and direct cellular interactions17. ACs, on the other hand, have been either directly isolated from explanted blood vessels or purified by FACS from human adipose tissue and skeletal muscle. A notable pro-angiogenic effect of ACs was demonstrated in a mouse hind-limb ischemia model19.&#160;Furthermore, ACs have also been shown to repair infarcted myocardium more efficiently than conventional MSCs, indicating the robust therapeutic potential of ACs in ischemic tissue repair20.
The current purification protocol grants simultaneous, prospective purification of MECs, PCs, and ACs from the vasculature of a single human skeletal muscle biopsy. This allows us to study and/or choose the optimal hBVSC subpopulation for distinct therapeutic purposes. Additionally, this new technique further expands the repertoire of stem/progenitor cells that can be derived from human skeletal muscle, making it an ideal source of multipotent precursor cells for regenerative medicine.

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 1</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C5894</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 2</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C1764</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 4</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C1889</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD34 APC</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:148px;">555824</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD45 APC-Cy7</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:148px;">557833</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD56 PE-Cy7</td>
			<td style="width:99px;">BD Pharmingen&#160;</td>
			<td style="width:148px;">557747</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD144 PE</td>
			<td style="width:99px;">Beckman Coulter</td>
			<td style="width:148px;">A07481</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Anti-human CD146 FITC</td>
			<td style="width:99px;">AbD Serotec</td>
			<td style="width:148px;">MCA2141F</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">FACSAria II Flow Cytometer</td>
			<td style="width:99px;">Becton-Dickinson</td>
			<td style="width:148px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">EGM-2 Complete Medium</td>
			<td style="width:99px;">Lonza</td>
			<td style="width:148px;">CC-3162</td>
			<td style="width:193px;">For culturing PCs (only P0)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:257px;">DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">11965</td>
			<td style="width:193px;">For culturing PCs</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">DMEM high glucose (1X), liquid, with L-glutamine, with sodium pyruvate</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">11995</td>
			<td style="width:193px;">For culturing MECs and ACs</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Fetal Bovine Serum</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">10437-028</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Heat-inactivated horse serum</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">26050-088</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Penicillin/Streptomycin</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15140-122</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Antibiotic-Antimycotic (100X)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15240-062</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Trypsin-EDTA 0.5%(10X)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15400-054</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Dulbecco&#8217;s PBS without calcium and magnesium</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">14190-250</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Chick embryo extract</td>
			<td style="width:99px;">Accurate Chemical</td>
			<td style="width:148px;">CE650T-10</td>
			<td style="width:193px;">Filter before use</td>
		</tr>
	</tbody>
</table>

isolation of blood-vessel-derived multipotent precursors from human skeletal muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

FACS parameters are first corrected based on the data obtained from the unstained control, negative controls, and single-color positive controls. After exclusion of dead cells, fluorescence-labeled cell suspension is subjected to a series of negative and positive cell surface marker selections. First, CD45+ cells are gated out before CD56+ and CD56- cells are separated from the CD45- fraction. The CD56+ fraction is further subjected to CD34-CD144 selection where only...

Watch this Scientific Journal Video about Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle at JoVE.com

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

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11.6K Views.  University of Pittsburgh. Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Video: Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...