This work was supported by Grants-in-Aid for Scientific Research (C) (grant number: 25461644) and Young Scientists (B) (grant numbers: 15K19614, 26860845, 17K16298) of JSPS KAKENHI.

The use of human samples for this study was approved by the ethical committee of Kobe University Graduate School of Medicine (approval no. 1370 and 1694) and conducted in accordance with the approved guidelines.
1. Isolation and Culture of UC-MSCs
NOTE: UC-MSCs have been successfully isolated, cultured, and expanded (more than passage number 4) from more than 200 UCs subjected to this protocol. Among more than 200 UCs, 100% have shown successful UC-MSC isolation, less than 5% have shown accidental contamination, less than 15% have shown growth arrest, and more than 80% have shown successful UC-MSC expansion.
<ol>
	<li>Collect UC.
	<ol>
		<li>Prepare a 50 mL plastic tube, an aseptic scissor, and a 500 mL bottle of alpha modified Eagle&#39;s minimum essential medium (alpha MEM) at 4 &#176;C.</li>
		<li>Aseptically cut out UC with a surgical scissor at approximately 5-10 cm in length and collect it soon after newborn birth by cesarean section.</li>
		<li>Immediately place the UC in a 50 mL plastic tube and add 20-30 mL of alpha MEM into the tube.</li>
		<li>Store the UC at room temperature (RT) until it is transported to the laboratory. 
		NOTE: Aseptic UC can be stored in serum-free medium at RT for up to 2 days. Therefore, UC obtained from neighboring hospitals can be collected if it is delivered to the lab within 2 days.</li>
	</ol>
	</li>
	<li>Dissect UC.
	<ol>
		<li>Prepare a plastic tray, sterilized scissors and forceps, 10 mL pipettes, 25 mL pipettes, two 60 mm tissue culture dishes, phosphate-buffered saline (PBS), purified enzyme blends (see Table of Materials, reconstituted with sterile PBS in a concentration of 13 W&#252;nsch units/mL and stored at -80 &#176;C), and a 500 mL bottle of reduced serum medium (see Table of Materials).</li>
		<li>Warm up the PBS, purified enzyme blends, reduced serum medium, and culture medium [alpha MEM containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (AA) stored at 4 &#176;C] to RT.</li>
		<li>Take out the UC from the tube and place it in a plastic tray.</li>
		<li>Weigh and dissect 5 g of UC with a sterilized scissor.</li>
		<li>Pour 10 mL of 70% ethanol over the UC for sterilization using a 10 mL pipette.</li>
		<li>Wash the UC with 10 mL of PBS twice using a 10 mL pipette.</li>
		<li>Place the UC in a sterilized 60 mm tissue culture dish (see Figure 1, step 1).</li>
		<li>Add 10 mL of reduced serum medium into the dish.</li>
		<li>Add 0.5 mL of purified enzyme blends to reach a final concentration of approximately 0.62 W&#252;nsch units/mL. 
		NOTE: The use of purified enzyme blends instead of traditional collagenase has been shown to improve the yield and viability of UC-MSCs isolated from UC.</li>
		<li>Cut the UC into 2-3 mm pieces with sterilized scissors and forceps (see Figure 1, step 2). 
		NOTE: This process takes about 30 min.</li>
		<li>Incubate the UC pieces at 37 &#176;C for 30 min in a 5% CO2 incubator.</li>
		<li>Cut the partially-digested UC pieces into smaller pieces that easily flow through a 25 mL pipette. 
		NOTE: This process takes about 30 min.</li>
		<li>Incubate the smaller UC pieces at 37 &#176;C for 15-45 min in a 5% CO2 incubator. 
		NOTE: Incubation needs to continue until the homogenates become viscous. The total digestion time is approximately 120 min. In the case of using UCs from preterm infants, however, the digestion time can be shortened because those are easily cut and digested.</li>
	</ol>
	</li>
	<li>Isolate UC-MSCs.
	<ol>
		<li>Prepare four 50 mL plastic tubes and a 500 mL bottle of culture medium.</li>
		<li>Divide the UC homogenate into two 50 mL plastic tubes using a 25 mL pipette, with approximately 7.5 mL in each tube.</li>
		<li>Add 20 mL of culture medium into each tube and mix well.</li>
		<li>Filter each UC homogenate through a 100 &#181;m cell strainer placed on top of a new 50 mL collection tube, using a 25 mL pipette to collect UC-derived cells. 
		NOTE: This process takes about 15 min each, as the digested UC solution is sticky.</li>
		<li>Centrifuge two tubes at 1,000 x g for 5 min.</li>
		<li>Carefully aspirate the supernatant and discard it.</li>
		<li>Resuspend the cell pellets in two tubes with 5 mL of culture medium.</li>
		<li>Transfer the cell suspension into a new 60 mm plate and culture at 37 &#176;C in a 5% CO2 incubator (see Figure 1, step 3).</li>
	</ol>
	</li>
	<li>Culture UC-MSCs.
	<ol>
		<li>Warm up the PBS, trypsin-EDTA (0.25 w/v%), and culture medium (alpha MEM containing 10% FBS and 1% AA) to RT.</li>
		<li>When cells are attached to a 60 mm plate, remove the culture medium and wash with 5 mL of PBS twice to remove debris and red blood cells. 
		NOTE: Attached cells usually appear at 3-5 days after initial plating (see Figure 1, step 4).</li>
		<li>Replace the culture medium twice per week and incubate at 37 &#176;C in a 5% CO2 incubator until 90-100% confluency. 
		NOTE: This usually takes 7-14 days after initial plating.</li>
		<li>Wash cells with 5 mL of PBS twice, add 0.5 mL of trypsin-EDTA, and incubate at 37 &#176;C for 5-10 min.</li>
		<li>When cells become rounded and detached, add 9 mL of culture medium and mix well to inactivate trypsin.</li>
		<li>Transfer the cell suspension into a new 100 mm plate and culture at 37 &#176;C in a 5% CO2 incubator (see Figure 1, step 5).</li>
		<li>Replace the culture medium twice per week and incubate at 37 &#176;C in a 5% CO2 incubator until 90-100% confluency. 
		NOTE: This usually takes 4-8 days after initial plating.</li>
		<li>Wash cells with 10 mL of PBS twice, add 1 mL of trypsin-EDTA, and incubate at 37 &#176;C for 5-10 min.</li>
		<li>When cells become rounded and detached, add 9 mL of culture medium and mix well to inactivate trypsin.</li>
		<li>Transfer the cell suspension into two new 100 mm plates and culture at 37 &#176;C in a 5% CO2 incubator (see Figure 1, step 5).</li>
		<li>Repeat subculture (1:2 splits) until tenth passage (see Figure 1, step 5). 
		NOTE: Because cells cultured under less confluent conditions tend to reach earlier proliferation arrest, it is important to passage cells with a split ratio of 1:2.</li>
		<li>Use cells at fifth to eighth passage for cell surface marker analysis, cell differentiation assay, and other experiments. 
		NOTE: To keep the vigorous proliferation for as long as possible, cells are generally cultured under more than 70-80% confluent conditions.</li>
	</ol>
	</li>
</ol>
2. Surface Marker Expression of UC-MSCs
<ol>
	<li>Prepare cells in a 100 mm plate and dissociate them with 1 mL of trypsin-EDTA at 37 &#176;C for 5-10 min.</li>
	<li>Add 9 mL of culture medium and centrifuge at 1,000 x g for 5 min.</li>
	<li>Aspirate the supernatant and discard it.</li>
	<li>Resuspend the cell pellets with 10 mL of PBS and centrifuge at 1,000 x g for 5 min. Repeat this washing step twice.</li>
	<li>Resuspend cells with flow cytometry (FCM) buffer (PBS containing 2 mM EDTA and 10% blocking reagent) at ~1 &#215; 106 cells/mL.</li>
	<li>Transfer 50-100 &#181;L of cell suspension into a 1.5 mL tube; add phycoerythrin (PE)-conjugated antibodies against CD14, CD19, CD34, CD45, CD73, CD90, CD105, or HLA-DR; and incubate on ice for 45 min.</li>
	<li>Wash cells with FCM buffer twice and add 50-100 &#181;L of 0.2% viability dye solution (see Table of Materials).</li>
	<li>Incubate at RT for 15 min, wash cells with FCM buffer twice, and filter cells through a 70 &#181;m cell strainer.</li>
	<li>Run cells through the FCM and analyze the obtained results using FCM software according to the manufacturer&#39;s instructions.</li>
</ol>
3. Trilineage Differentiation of UC-MSCs
<ol>
	<li>Perform adipogenesis.
	<ol>
		<li>Prepare a cell suspension in culture medium at a concentration of 5 &#215; 103 cells/mL.</li>
		<li>Plate 1 mL of cell suspension in a 12-well plate and incubate at 37 &#176;C in a 5% CO2 incubator for ~24 h.</li>
		<li>Replace the culture medium with adipogenic differentiation medium (see Table of Materials) and incubate at 37 &#176;C in a 5% CO2&#160;incubator for 2-3 weeks. Change adipogenic differentiation medium twice a week.</li>
		<li>Wash cells with PBS three times.</li>
		<li>Incubate cells in 4% formaldehyde solution for 30 min at RT.</li>
		<li>Wash cells with PBS three times and with 60% isopropanol three times.</li>
		<li>Prepare 0.5% oil red O solution by dissolving 84 mg of oil red O in 10 mL of 100% isopropanol and adding 6.7 mL of distilled water. Stain cells with 1 mL of 0.5% oil red O solution at RT for 20 min.</li>
		<li>Wash cells with 60% isopropanol three times and visualize under a microscope.</li>
	</ol>
	</li>
	<li>Perform osteogenesis.
	<ol>
		<li>Prepare a cell suspension in culture medium at a concentration of 1 &#215; 104 cells/mL.</li>
		<li>Plate 1 mL of cell suspension in a 12-well plate and incubate at 37 &#176;C in a 5% CO2 incubator for ~24 h.</li>
		<li>Replace the culture medium with osteogeneic differentiation medium (see Table of Materials) and incubate at 37 &#176;C in a 5% CO2 incubator for 1-2 weeks. Change osteogenic differentiation medium twice a week.</li>
		<li>Wash cells with PBS three times.</li>
		<li>Incubate cells in 4% formaldehyde solution for 30 min at RT.</li>
		<li>Wash cells with PBS three times.</li>
		<li>Prepare 2% alizarin red S solution by dissolving 200 mg of alizarin red S with 10 mL of distilled water. Stain cells with 1 mL of 2% alizarin red S solution at RT for 3 min.</li>
		<li>Wash wells with PBS three times and visualize under a microscope.</li>
	</ol>
	</li>
	<li>Perform chondrogenesis.
	<ol>
		<li>Prepare a cell suspension in culture medium at a concentration of 1.6 &#215; 107 cells/mL.</li>
		<li>Place a 5 &#181;L droplet of cell suspension onto the center of well in a 12-well plate (micromass cultures) and incubate at 37 &#176;C in a 5% CO2 incubator for 2-3 h.</li>
		<li>Gently add 1 mL of chondrogenic differentiation medium (see Table of Materials) and incubate at 37 &#176;C in a 5% CO2 incubator for 4-7 days. Change chondrogenic differentiation medium twice a week.</li>
		<li>Wash cells with PBS three times.</li>
		<li>Incubate cells in 4% formaldehyde solution for 30 min at RT.</li>
		<li>Wash cells with PBS three times.</li>
		<li>Prepare 0.05% toluidine blue solution by dissolving 250 mg of toluidine blue with 500 mL of 0.1 M sodium acetate buffer with pH 4.1. Stain cells with 1 mL of 0.05% toluidine blue solution at RT for 30 min.</li>
		<li>Wash cells with PBS three times and visualize under a microscope.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

MSCs can be isolated from a variety of tissues and are heterogeneous population of cells that do not all express the same phenotypic markers. Here, we outlined a protocol that guides the collection and dissection of UC from preterm and term infants and enables isolation and culture of UC-MSCs. Following this protocol, we have successfully isolated UC-MSCs that fulfill the ISCT criteria19 from fetuses/infants delivered at 19-40 weeks of gestation and demonstrated that they represent some aspects of...

Mesenchymal stem cells (MSCs) are originally isolated and characterized from bone marrow (BM)1,2 but can also be obtained from a wide variety of tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages3. MSCs are recognized as a heterogeneous cell population that can proliferate and differentiate into adipocytes, osteocytes, and chondrocytes. In addition, MSCs possess the ability to migrate to sites of injury, suppress and modulate immune responses, and remodel and repair injury. Currently, MSCs from different sources have attracted growing interest as a source for cell therapy against a number of intractable diseases, including graft-versus-host disease, myocardial infarction, and cerebral infarction4,5.
Although BM is the most well-characterized source of MSCs, the invasiveness of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC) have advantages including less ethical concerns regarding sampling and robust proliferation and differentiation capacity6,7. Among fetal appendages that are usually discarded as medical waste, UCB and UC are considered a fetal organ, while placenta is considered fetomaternal. In addition, placenta and UCB need to be sampled and collected at the exact moment of newborn delivery, whereas placenta and UC can be collected and processed after newborn delivery. Accordingly, UC is a promising MSC source for cell therapy8,9.
Human UC starts to develop with progressive expansion of the amniotic cavity at 4-8 weeks of gestation, continues to grow until 50-60 cm in length, and can be isolated during the whole period of newborn delivery10. To gain insight into the pathophysiology of intractable diseases, we use UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages11,12. In this protocol, we describe how to isolate and characterize UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

<table><tbody><tr><td>50 mL plastic tube</td><td>AS One Coporation, Osaka, Japan</td><td>Violamo 1-3500-22</td><td></td></tr><tr><td>12-well plate</td><td>AGC Techno Glass, Tokyo, Japan</td><td>Iwaki 3815-012</td><td></td></tr><tr><td>60 mm dish</td><td>AGC Techno Glass, Tokyo, Japan</td><td>Iwaki 3010-060</td><td></td></tr><tr><td>Cell strainer (100 &amp;mu;m)</td><td>Thermo Fisher Scientific, Waltham, MA&amp;nbsp;</td><td>Falcon 35-2360</td><td></td></tr><tr><td>Cell strainer (70 &amp;mu;m)</td><td>Thermo Fisher Scientific, Waltham, MA&amp;nbsp;</td><td>Falcon 35-2350</td><td></td></tr><tr><td>Alpha MEM</td><td>Wako Pure Chemical, Osaka, Japan</td><td>135-15175</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma Aldrich, St. Louis, MO</td><td>172012</td><td></td></tr><tr><td>Reduced serum medium</td><td>Thermo Fisher Scientific, waltham, MA</td><td>OPTI-MEM Gibco 31985-070</td><td></td></tr><tr><td>Antibiotic-antimycotic</td><td>Thermo Fisher Scientific, Waltham, MA&amp;nbsp;</td><td>Gibco 15240-062</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Wako Pure Chemical, Osaka, Japan</td><td>209-16941</td><td></td></tr><tr><td>PBS</td><td>Takara BIO, Shiga,Japan</td><td>T900</td><td></td></tr><tr><td>Purified enzyme blends</td><td>Roche, Mannheim, Germany</td><td>Liberase DH Research Grade 05401054001</td><td></td></tr><tr><td>PE-conjugated mouse primary antibody against CD14</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>347497</td><td>Lot: 3220644, RRID: AB_400312</td></tr><tr><td>PE-conjugated mouse primary antibody against CD19</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>340364</td><td>Lot: 3198741, RRID: AB_400018</td></tr><tr><td>PE-conjugated mouse primary antibody against CD34</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>555822</td><td>Lot: 3079912, RRID: AB_396151</td></tr><tr><td>PE-conjugated mouse primary antibody against CD45</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>555483</td><td>Lot: 2300520, RRID: AB_395875</td></tr><tr><td>PE-conjugated mouse primary antibody against CD73</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>550257</td><td>Lot: 3057778, RRID: AB_393561</td></tr><tr><td>PE-conjugated mouse primary antibody against CD90</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>555596</td><td>Lot: 3128616, RRID: AB_395970</td></tr><tr><td>PE-conjugated mouse primary antibody against CD105</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>560839</td><td>Lot: 4339624, RRID: AB_2033932</td></tr><tr><td>PE-conjugated mouse primary antibody against HLA-DR</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>347367</td><td>Lot: 3219843, RRID: AB_400293</td></tr><tr><td>PE-conjugated mouse IgG1 k isotype</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>555749</td><td>Lot: 3046675, RRID: AB_396091</td></tr><tr><td>PE-conjugated mouse IgG2a k isotype</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>555574</td><td>Lot: 3035934, RRID: AB_395953</td></tr><tr><td>PE-conjugated mouse IgG2b k isotype</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>555743</td><td>Lot: 3098896, RRID: AB_396086</td></tr><tr><td>Viability dye</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>Fixable Viability Stain 450 562247</td><td></td></tr><tr><td>Blocking reagent</td><td>Dainippon Pharmaceutical, Osaka, Japan</td><td>Block Ace UKB80</td><td></td></tr><tr><td>FCM</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>BD FACSAria&amp;nbsp; III Cell Sorter</td><td></td></tr><tr><td>FCM software</td><td>BD Bioscience, Franklin Lakes, NJ</td><td>BD FACSDiva</td><td></td></tr><tr><td>Adipogenic differentiation medium</td><td>Invitrogen, Carlsbad, CA</td><td>StemPro Adipogenesis Differentiation kit A10070-01</td><td></td></tr><tr><td>Osteogenic differentiation medium</td><td>Invitrogen, Carlsbad, CA</td><td>StemPro Osteogenesis Differentiation kit A10072-01</td><td></td></tr><tr><td>Chondrogenic differentiation medium&amp;nbsp;</td><td>Invitrogen, Carlsbad, CA</td><td>StemPro Chondrogenesis Differentiation kit A10071-01</td><td></td></tr><tr><td>Formaldehyde</td><td>Polyscience, Warrigton, PA</td><td>16% UltraPure Formaldehyde EM Grade #18814</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma Aldrich, St. Louis, MO</td><td>O0625</td><td></td></tr><tr><td>Arizarin Red S</td><td>Sigma Aldrich, St. Louis, MO</td><td>A5533</td><td></td></tr><tr><td>Toluidine Blue</td><td>Sigma Aldrich, St. Louis, MO</td><td>198161</td><td></td></tr><tr><td>Microscope</td><td>Keyence, Osaka, Japan</td><td>BZ-X700</td><td></td></tr></tbody></table>

isolation and characterization of human umbilical cord-derived mesenchymal stem cells from preterm and term infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

The procedures from UC collection to MSC culture are summarized in Figure 1. UC of approximately 5-10 cm in length can be collected from all newborns delivered by cesarean section. UC starts to develop at 4-8 weeks of gestation and continues to grow until 50-60 cm in length, as shown in Figure 2. There are two arteries (A), one vein (V), cord lining (CL), and Wharton&#39;s Jelly (WJ) in UC, as depicted in Fig...

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Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

isolation-characterization-human-umbilical-cord-derived-mesenchymal

Research

JoVE Journal

Developmental Biology

14.0K Views.  Kobe University Graduate School of Medicine. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de beraadslaging van UCMSC, zoals de beste bron van USMSC voor celtherapie. Het belangrijkste voordeel van deze techniek is dat het een consistente isolatie en krachtige proliferatie van UCMSC van pre-term en term zuigelingen mogelijk maakt. De implicaties van deze techniek breiden zich uit tot therapie van hardnekkige ziekten.Hoewel deze methode inzicht kan geven in de beraadslaging van UCMSC, kan het ook worden to...

Video: Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Biology

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the reliability and robustness of the source. We describe here a stepwise protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue (uMSCs), their immunophenotyping, and the propagation of such cultures over several passages. In this procedure, the viability of the uMSCs is high because there is no enzymatic digestion. Further, the removal of blood vessels, including the umbilical cord arteries and the vein, ensures that there is no contamination of cells of endothelial origin. Using flow cytometry, uMSCs upon isolation are CD45&#8722;CD34&#8722;, indicating an absence of cells from the hematopoietic lineage. Importantly, they express key surface markers, CD105, CD90, and CD73. Upon establishment of cultures, this paper describes an efficient method to induce differentiation in these uMSCs into the skeletal muscle lineage. A detailed analysis of myogenic progression in differentiated uMSCs reveals that uMSCs express Pax7, a marker for myogenic progenitors in the initial stages of differentiation, followed by the expression of MyoD and Myf5, and, finally, a terminal differentiation marker, myosin heavy chain (MyHC).

We describe a protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue and their differentiation into the skeletal muscle lineage.

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the ...

Isolation and Culture of Three Kinds of Umbilical Cord Mesenchymal Stem Cells

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord Wharton's jelly, as well as from the umbilical arteries and umbilical vein. They are known as perivascular stem cells obtained from umbilical arteries (UCA-PSCs), perivascular stem cells obtained from the umbilical vein (UCV-PSCs), and mesenchymal stem cells obtained from Wharton's jelly (WJ-MSCs). UCA-PSCs and UCV-PSCs are pericytes derived from perivascular regions that are progenitors of MSCs. Isolation and culture of the three kinds of cells is an important source for studying stem cell transplantation and repair. The present protocol focuses on the isolation and culture of cells through mechanical separation, adherent culture, and cell crawling out. Through this technique, the three different types of stem cells can be derived. Cell surface markers were detected by flow cytometry. The stem cells were detected for multilineage differentiation potential by adipogenic, osteogenic, and neural-like differentiation, which is consistent with the phenotype of MSCs. This experimental protocol expands the source of UC-MSCs. In addition, the cell isolation method provides a basis for further study of regenerative medicine and other applications.

The present protocol describes the isolation and culture of mesenchymal stem cells from the umbilical cord arteries, vein, and Wharton's jelly.

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord ...

Dissection and Isolation of WJ-MSCs from the Human Umbilical Cord

Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) from the umbilical cord (UC) has gained increasing interest in the biomedical field as an outstanding source of MSCs. However, challenges such as limited supply and lack of standardization in existing methods have arisen. This article presents a novel method for enhancing MSC yield by dissecting intact WJ from the umbilical cord. The method employs blunt dissection to remove the epithelial layer, maintaining the integrity of the entire WJ and resulting in an increased quantity and viability of harvested MSCs. This approach significantly reduces WJ waste compared to conventional sharp dissection methods. To ensure the purity of WJ-MSCs and minimize external cellular influence, a procedure utilizing internal tension to peel off the endothelium after flipping the UC was conducted. Additionally, the Petri dish was inverted for a short time during explant culture to improve attachment and cell outgrowth. Comparative analysis demonstrated the superiority of the proposed method, showing a higher yield of WJ and WJ-MSCs with better viability than traditional methods. The similar morphology and expression pattern of cell surface markers in both methods confirm their characterization and purity for various applications. This method provides a high-yield and high-viability approach for WJ-MSC isolation, demonstrating great potential for the clinical application of MSCs.

Author Spotlight: Advancements in Stem Cell-Derived Vascularized Organoids and Optimized Isolation of Wharton's Jelly for Enhanced MSC Production

This study presents a unique blunt dissection procedure to preserve the integrity of Wharton's jelly (WJ), resulting in less damaged WJ and a greater quantity and viability of the harvested mesenchymal stem cells (MSCs). The method demonstrates superior yield and proliferative ability compared to conventional sharp dissection methods.

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) ...

Medicine

Functional Analysis of BAMBIhighMFGE8high UC-MSC Subpopulations

Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIhighMFGE8high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIhighMFGE8high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIhighMFGE8high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

The isolation of BAMBIhighMFGE8high MSCs, one of the three main subgroups constituting heterogeneous human UC-MSCs, is helpful for fully understanding the characteristics and functions of this subtype for its future application to improve clinical efficacy in specific diseases. Here, we present a method for sorting BAMBIhighMFGE8high UC-MSCs.

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

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