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-MS...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" style="width:1455px;" width="1455">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">50mL plastic tube</td>
			<td style="width:336px;">AS One Coporation, Osaka, Japan</td>
			<td style="width:379px;">Violamo 1-3500-22</td>
			<td style="width:312px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">12-well plate</td>
			<td>AGC Techno Glass, Tokyo, Japan</td>
			<td style="width:379px;">Iwaki 3815-012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">60mm dish</td>
			<td>AGC Techno Glass, Tokyo, Japan</td>
			<td style="width:379px;">Iwaki 3010-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Cell strainer (100 &#956;m)</td>
			<td style="width:336px;">Thermo Fisher Scientific, Waltham, MA&#160;</td>
			<td style="width:379px;">Falcon 35-2360</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Cell strainer (70 &#956;m)</td>
			<td style="width:336px;">Thermo Fisher Scientific, Waltham, MA&#160;</td>
			<td style="width:379px;">Falcon 35-2350</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Alpha MEM</td>
			<td style="width:336px;">Wako Pure Chemical, Osaka, Japan</td>
			<td style="width:379px;">135-15175</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Fetal bovine serum</td>
			<td style="width:336px;">Sigma Aldrich, St. Louis, MO</td>
			<td style="width:379px;">172012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Reduced serum medium</td>
			<td style="width:336px;">Thermo Fisher Scientific, waltham, MA</td>
			<td style="width:379px;">OPTI-MEM Gibco 31985-070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Antibiotic-antimycotic</td>
			<td style="width:336px;">Thermo Fisher Scientific, Waltham, MA&#160;</td>
			<td style="width:379px;">Gibco 15240-062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Trypsin-EDTA</td>
			<td style="width:336px;">Wako Pure Chemical, Osaka, Japan</td>
			<td style="width:379px;">209-16941</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PBS</td>
			<td style="width:336px;">Takara BIO, Shiga,Japan</td>
			<td style="width:379px;">T900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Purified enzyme blends</td>
			<td style="width:336px;">Roche, Mannheim, Germany</td>
			<td style="width:379px;">Liberase DH Research Grade 05401054001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse primary antibody against CD14</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">347497</td>
			<td>Lot: 3220644, RRID: AB_400312</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse primary antibody against CD19</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">340364</td>
			<td>Lot: 3198741, RRID: AB_400018</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse primary antibody against CD34</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">555822</td>
			<td>Lot: 3079912, RRID: AB_396151</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse primary antibody against CD45</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">555483</td>
			<td>Lot: 2300520, RRID: AB_395875</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse primary antibody against CD73</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">550257</td>
			<td>Lot: 3057778, RRID: AB_393561</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse primary antibody against CD90</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">555596</td>
			<td>Lot: 3128616, RRID: AB_395970</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse primary antibody against CD105</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">560839</td>
			<td>Lot: 4339624, RRID: AB_2033932</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse primary antibody against HLA-DR</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">347367</td>
			<td>Lot: 3219843, RRID: AB_400293</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse IgG1 k isotype</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">555749</td>
			<td>Lot: 3046675, RRID: AB_396091</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse IgG2a k isotype</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">555574</td>
			<td>Lot: 3035934, RRID: AB_395953</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">PE-conjugated mouse IgG2b k isotype</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">555743</td>
			<td>Lot: 3098896, RRID: AB_396086</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Viability dye</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td style="width:379px;">Fixable Viability Stain 450 562247</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Blocking reagent</td>
			<td style="width:336px;">Dainippon Pharmaceutical, Osaka, Japan</td>
			<td style="width:379px;">Block Ace UKB80</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">FCM</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td>BD FACSAria&#160; III Cell Sorter</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">FCM software</td>
			<td style="width:336px;">BD Bioscience, Franklin Lakes, NJ</td>
			<td>BD FACSDiva</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Adipogenic differentiation medium</td>
			<td style="width:336px;">Invitrogen, Carlsbad, CA</td>
			<td>StemPro Adipogenesis Differentiation kit A10070-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Osteogenic differentiation medium</td>
			<td style="width:336px;">Invitrogen, Carlsbad, CA</td>
			<td>StemPro Osteogenesis Differentiation kit A10072-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chondrogenic differentiation medium&#160;</td>
			<td style="width:336px;">Invitrogen, Carlsbad, CA</td>
			<td>StemPro Chondrogenesis Differentiation kit A10071-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formaldehyde</td>
			<td style="width:336px;">Polyscience, Warrigton, PA</td>
			<td>16% UltraPure Formaldehyde EM Grade #18814</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Oil Red O</td>
			<td style="width:336px;">Sigma Aldrich, St. Louis, MO</td>
			<td style="width:379px;">O0625</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Arizarin Red S</td>
			<td style="width:336px;">Sigma Aldrich, St. Louis, MO</td>
			<td style="width:379px;">A5533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Toluidine Blue</td>
			<td style="width:336px;">Sigma Aldrich, St. Louis, MO</td>
			<td style="width:379px;">198161</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:428px;">Microscope</td>
			<td style="width:336px;">Keyence, Osaka, Japan</td>
			<td style="width:379px;">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...

Watch this Scientific Journal Video about Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants at JoVE.com

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 ...

This method can help answer key questions in the deliberation of UCMSC, such as the best source of USMSC for cell therapy. The main advantage of this technique is that it enables consistent isolation and vigorous proliferation of UCMSC from pre-term and term infants. The implications of this technique extend toward therapy of intractable diseases.Though this method can provide insight into the deliberation of UCMSC, it can also be applied to other MSC sources, such as adipose tissue, synovium, skin, dental pulp, and so on. Generally, an individual new to this method will struggle because there are so many variation in the steps of umbilical dissection and mesenchymal stem cell isolation. Visual demonstration of this method is critical as these steps are difficult to learn because there are several points prone to be overlooked.To begin dissecting the umbilical cord, first prepare a metal tray, sterilized scissors, and forceps, ten milliliter pipettes, 25 milliliter pipettes, and two 60 millimeter tissue culture dishes. Warm up PBS purified enzyme blends, a 500 milliliter of reduced serum medium and culture medium, to room temperature. Remove previously isolated umbilical cord from a 50 milliliter tube in alpha MEM, and place it in the plastic tray.Weigh the umbilical cord, and then use sterilized scissors to dissect five grams of the cord. To sterilize the umbilical cord, use ten milliliter pipette to pour ten milliliters of 70 percent ethanol over it. Then wash the cord twice by adding ten milliliters of PBS with ten milliliter pipette.Place the washed umbilical cord in the sterilized 60 millimeter tissue culture dish and add ten milliliters reduced serum medium and 0.5 milliliters purified enzyme blends. Use sterilized scissors and forceps to cut the cord into two to three millimeter pieces. Place the dish in a five percent carbon dioxide incubator and incubate at 37 degrees Celsius for 30 minutes.Then cut the partially digested pieces into smaller pieces, and incubate them at 37 degrees Celsius in a five percent carbon dioxide incubator until the homogenate becomes viscous. Finally, make sure that the homogenate flows easily through a 25 milliliter pipette. First prepare four, 50 milliliter plastic tubes in a 500 milliliter bottle of culture medium.Use a 25 milliliter pipette to divide the umbilical cord homogenate into two, 50 milliliter plastic tubes with approximately 7.5 milliliters in each tube. Then add 20 milliliters of culture medium into each tube and mix well. Collect each homogenate with 25 milliliter pipette and filter through a 100 micrometer cell strainer placed on top of a new 50 milliliter collection tube.Centrifuge two tubes with filtrates at 1000g for five minutes at room temperature. After finished centrifugation, carefully aspirate the supernatant and discard it. Add five milliliters of culture medium to each tube, and re-suspend the cell pellets.Transfer the cell suspension into a new 60 millimeter plate and culture at 37 degrees Celsius in a five percent carbon dioxide incubator. To culture the UCMSC's, warm the PBS, trypsin EDTA, and culture medium, to room temperature. When cells are attached to the plate, remove the medium, and wash twice with five milliliters of PBS to remove debris and red blood cells.Add the culture medium and incubate at 37 degrees Celsius in a five percent carbon dioxide incubator. Replace the medium twice per week, and culture the cells until they reach 90 to 100 percent confluency. Then wash the cells twice with five milliliters of PBS, add 0.5 milliliters of trypsin EDTA, and incubate at 37 degrees Celsius for five to ten minutes.When cells become rounded and detached, add nine milliliters of culture medium, and mix well to inactivate trypsin. Add the cell suspension into a new 100 millimeter plate. Grow the cells at 37 degrees Celsius in a five percent carbon dioxide incubator until they reach 90 to 100 percent confluency.Repeat trypsinization and dividing into two new plates, until the tenth passage. Surface marker expression of UCMSC's was used as criterion for their characterization. When incubated with appropriate antibodies and analyzed by flow cytometry, they were found positive for MSC's signature markers, but negative for monocyte macrophage, negative for hematapoietic stem cell and ephithelial cell, negative for B cell, negative for pan leukocytes, and negative for major histocompatibility complex markers.To evaluate trilineage differentiation capacity of USCMSC's from preterm and term infants, the cells were induced to differentiate into adipocytes, osteocytes, and chondrocytes. UCMSC's successfully differentiated into all three types of mesodermal cells confirming their differentiation capacity. While attempting this procedure, it's important to remember that there are four critical steps.Cutting the cord into two to three centimeter pieces, cutting into smaller pieces, making sure that the homogenate flows easily through a 25 milliliter pipette, and filtering the homogenate through a 100 micrometer cell strainer.

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13.6K Views.  Kobe University Graduate School of Medicine. This method can help answer key questions in the deliberation of UCMSC, such as the best source of USMSC for cell therapy. The main advantage of this technique is that it enables consistent isolation and vigorous proliferation of UCMSC from pre-term and term infants. The implications of this technique extend toward therapy of intractable diseases.Though this method can provide insight into the deliberation of UCMSC, it can also be applied to other MSC sources, such as adipose tissue, s...