We acknowledge the scholarship and financial support&#160;&#160;provided by the Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACyT No.49887),&#160;Facultad de Medicina-UNAM DGAPA IN201510 and DGAPA IN216213 ; Salvador Correa of Instituto Nacional de Perinatolog&#237;a for help with the biological material; and Biocriss, S. A. de C. V for donating Nukbone. Also thank to Ing. H&#233;ctor Mart&#237;nez of IIM, UNAM and M en C Fabiola &#160;Gonzalez of CINVESTAV for technical&#160;assistance.&#160;

This research was performed in accordance with the World Medical Association&#8217;s Declaration of Helsinki regarding the ethical conduct of research involving humans and was approved by the Research Ethic Board of Facultad de Medicina, UNAM (project number 101-2012).
1. Isolation of Human Mesenchymal Stem Cell
<ol>
	<li>Preparation of reagent
	<ol>
		<li>Prepare phosphate-buffered saline solution (PBS) and supplement with 1% antibiotic-antimycotic solution.</li>
		<li>Prepare Dulbecco&#39;s Modified Eagle&#39;s Medium, high glucose (HG-DMEM), and add 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution.Sterilize this culture medium by filtration through a 0.2&#160;&#181;m filter.</li>
		<li>Prepare a collagenase II solution at 100 U/ml in HG-DMEM without FBS or antibiotic-antimycotic solution.</li>
		<li>Sterilize the following surgical instruments,which are needed for the isolation, by autoclaving: standard scissors, simple dissecting forceps, fine-point and toothed forceps and a scalpel handle (#4).</li>
	</ol>
	</li>
	<li>Isolation of mesenchymal stem cells of human amniotic membrane
	<ol>
		<li>Hold the umbilical cord with one hand and with the other hand, detach the amniotic membrane (AM), which looks like a translucent sheet, to obtain a fragment of approximately 5 cm2. If the chorion section is present in the sample, separate the underlying chorion by manual dissection as the chorion section is thicker than the amnion.</li>
		<li>Remove residual blood with three washes of 5 ml of PBS and remove clots with the simple forceps. Make a small cut in the AM with the scalpel to generate fragments of approximately 0.5 cm2.</li>
	</ol>
	</li>
	<li>Enzymatic digestion of amniotic membrane
	<ol>
		<li>Incubate the AM with 5 ml of 0.125% trypsin/0.5 mM EDTA solution at 37 &#176;C for 30 min in a 50 ml conical tube at 37 &#176;C in a 5% CO2 atmosphere. Centrifuge at 250 x g and 25 &#176;C for 5 min and then remove the trypsin solution by discarding the supernatant.</li>
		<li>Add 15 ml of collagenase type II solution and incubate for 2 hr at 37 &#176;C in a 5% CO2 atmosphere with occasional shaking according to Leyva et al.9</li>
		<li>Immediately after digestion with collagenase II, add 15 ml of PBS and centrifuge at 250 x g and 25 &#176;C for 10 min.</li>
		<li>Discard the supernatant and wash the cellular pellet with 15 ml of PBS solution and centrifuge at 250 x g and 25 &#176;C for 15 min. Discard the supernatant.</li>
		<li>Resuspend the cellular pellet in 10 ml of HG-DMEM by shaking gently.</li>
	</ol>
	</li>
	<li>Expansion of mesenchymal stem cells
	<ol>
		<li>Seed 2 ml of cell suspension (1 x 104 cells/cm2) in culture flask, and add 2 ml of HG-DMEM and incubate the cells at 37 &#176;C in a 5% CO2 atmosphere. Eliminate the non-adherent cell by changing the culture medium after 5-7 days.</li>
		<li>After this time, change the culture medium every 3 days for an additional 7-10 days to obtain a cellular confluence of 90%.</li>
		<li>Recover the cells and incubate them for 5 min at 37 &#176;C in a 5% CO2 atmosphere with 0.125% trypsin/EDTA solution (trypsinization).</li>
		<li>Immediately after trypsinization, collect cells with a pipette and transfer to a 15 ml conical tube and centrifuge at 250 x g for 5 min.</li>
		<li>Discard the supernatant and resuspend the cellular pellet in 10 ml of HG-DMEM.</li>
		<li>Culture cell suspension in two 75 cm2 culture flasks (5 ml in each flask), and maintain the culture until 90% confluence.</li>
		<li>Repeat steps 1.4.3. to 1.4.6, until the 9th passage or subculture.</li>
		<li>Recover cells by trypsinization as in steps 1.4.3 to 1.4.5 for flow cytometry or other studies.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Porous Bone Matrix Disk (NKB)
<ol>
	<li>To wet the NKB disks (diameter, 12 mm; thickness, 2 mm), incubate each disk overnight with 3 ml of HG-DMEM without FBS in a humidified atmosphere of 5% CO2 at 37 &#176;C, according to Fassina et al. 5</li>
	<li>After the wetting process, place the disk in a 50 ml conical tube and spin at 250 x g for 5 min to remove excess culture medium. Place the disk in a 24 well cell culture plate.
	<ol>
		<li>Add 500 &#181;l of HG-DMEM and incubate for 30 min at 25 &#176;C. Ensure that no bubbles are present on the NKB disk to favor the correct distribution of nutrients and cells.</li>
		<li>If bubbles are observed on the disk, shake and add 300 &#181;l of culture medium and incubate for 15 min in a humidified atmosphere of 5% CO2 at 37 &#176;C. After this time remove 300 &#181;l of culture medium sucking through the wall of well cell culture and confirm that no bubbles form.</li>
	</ol>
	</li>
	<li>Place the disk in a new 24-well plate using simple forceps.</li>
</ol>
3. Culture of Human Mesenchymal Stem Cells on the Porous Bovine Matrix
<ol>
	<li>Seed a cellular suspension (5 x 105 AM-hMSCs in 100 &#181;l of HG-DMEM) from three subcultures, on the surface of the pre-wetted biomaterial disk and incubate for 30 min in a humidified atmosphere of 5% CO2 at 37 &#176;C. Perform this step slowly and continuously on the upper face of the biomaterial to allow the cells to interact with the topography of all scaffold. Note: This procedure achieves the 60% of the cells attached on the biomaterial shown in the Table 1.</li>
	<li>Add 1.5 ml of HG-DMEM at 37 &#176;C, and maintain the culture for 3 days in a humidified atmosphere of 5% CO2 at 37 &#176;C. Do not generate turbulence in the addition of culture medium for avoid that the deposition of cells on the bottom of culture plate dish. This is initial time.</li>
</ol>
4. Cell Surface Markers Characterization by Flow Cytometry
<ol>
	<li>Detach the cells from 9th subculture before seeding them on the matrix disk. Use 0.25% trypsin/1 mM EDTA and fix them for 30 min in ice-cold 2% formaldehyde. Following fixation, wash the cells once with 0.05% PBS.</li>
	<li>Block non-specific binding by incubating the cells with 20% human immunoglobulin for 3 min on ice and incubate 0.1 x 106 cell aliquots with phycoerythrin (PerCP)-conjugated MAb against CD73, FITC-conjugated MAb CD90, PE-conjugated MAb against CD34, FITC-conjugated MAb CD45 and PE-conjugated MAb CD105 for 1 hr at 25 &#176;C in the dark. Isotype-matched FITC-conjugated MAb, PE-conjugated MAb andPerCP-conjugated MAb will serve as negative controls.</li>
	<li>Calculate the expression levels of the surface markers using software for data acquisition and analysis.</li>
</ol>
5. Cell Adhesion Detection by Scanning Electron Microscopy
<ol>
	<li>Rinse the cell-disk samples at 3 days of culture twice with PBS and fixthe samples with 4% (v/v) glutaraldehyde solutionin a 0.1 M Na-cacodylate buffer (pH 7.2).</li>
	<li>Prepare the samples for microscopic observation, as previously reported by Rivera-N et al.10 and observe using a scanning electron microscope at an electric potential of 15 kV and at a magnification of 500X and 2000X.</li>
</ol>
6. Cell Proliferation Assay
<ol>
	<li>To the cell-disk samples containing 1.5 ml of culture medium, add 150 &#181;l of vital colorant(AB) according to manufacturer&#8217;s guidelines and incubate the cells in a humidified atmosphere of 5% CO2 at 37 &#176;C.</li>
	<li>Immediately after the addition of AB (0 hr) and each 24 hr until reaching 7 days of cell culture, measure the absorbance at 570 nm with a reference wavelength of 600 nm.</li>
</ol>
7. Colony Forming Unit (CFU)11
<ol>
	<li>Culture 100 cells per 100 mm tissue culture diskin HG-DMEM and incubate for 14 days in a humidified atmosphere of 5% CO2 at 37 &#176;C.</li>
	<li>Wash the culture with PBS and stain with 0.5% crystal violet in methanol for 5-10 min at room temperature.</li>
	<li>Wash the plates with PBS twice and count the visible colonies using a hemocytometer.</li>
</ol>

<ol>
	<li>Alviano, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Term+amniotic+membrane+is+a+high+throughput+source+for+multipotent+mesenchymal+stem+cells+with+the+ability+to+differentiate+into+endothelial+cells+in+vitro.">Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells with the ability to differentiate into endothelial cells in vitro.</a> BMC Developmental Biology. 7, 1-14 (2007).</li><li>Anker, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mesenchymal+stem+cells+of+fetal+or+maternal+origin+from+human+placenta.">Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta.</a> Stem Cells. 22 (4), 1338-1345 (2004).</li><li>Seebach, C., Schultheiss, J., Wilhelm, K., Frank, J., Henrich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+six+bone-graft+substitutes+regarding+to+cell+seeding+efficiency,+metabolism+and+growth+behaviour+of+human+mesenchymal+stem+cells+(MSC)+in+vitro.">Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism and growth behaviour of human mesenchymal stem cells (MSC) in vitro.</a> Int J Care Injured. 41, 731-738 (2010).</li><li>Meseguer-Olmo, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+growth+kinematics+of+human+osteoblasts+on+porous+hydroxyapatite+ceramics.">In vitro growth kinematics of human osteoblasts on porous hydroxyapatite ceramics.</a> Rev. Ortop. Traumatol. 50, 224-232 (2006).</li><li>Fassina, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-power+ultrasound+as+a+tool+to+culture+human+osteoblasts+inside+cancellous+hydroxyapatite.">Low-power ultrasound as a tool to culture human osteoblasts inside cancellous hydroxyapatite.</a> Bioinorganic Chemistry and Applications. 2010, 1-8 (2010).</li><li>Fassina, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonic+and+Electromagnetic+Enhancement+of+a+Culture+of+Human+SAOS-2+Osteoblasts+Seeded+onto+a+Titanium+Plasma-Spray+Surface.">Ultrasonic and Electromagnetic Enhancement of a Culture of Human SAOS-2 Osteoblasts Seeded onto a Titanium Plasma-Spray Surface.</a> Tissue Engineering Part C: Methods. 15, 233-242 (2009).</li><li>Gomes, M. E., Holtorf, H. L., Reis, R. L., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+porosity+of+starch-based+fiber+mesh+scaffolds+on+the+proliferation+and+osteogenic+differentiation+of+bone+marrow+stromal+cells+cultured+in+a+flow+perfusion+bioreactor.">Influence of the porosity of starch-based fiber mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal cells cultured in a flow perfusion bioreactor.</a> Tissue Eng. 12, 801-809 (2006).</li><li>Rodriguez-Fuentes, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nukbone(R)+promotes+proliferation+and+osteoblastic+differentiation+of+mesenchymal+stem+cells+from+human+amniotic+membrane.">Nukbone(R) promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane.</a> Biochemical and Biophysical Research Communications. 434, 676-680 (2013).</li><li>Leyva-Leyva, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mesenchymal+stem+cell+subpopulations+from+human+amniotic+membrane+with+dissimilar+osteoblastic+potential.">Characterization of mesenchymal stem cell subpopulations from human amniotic membrane with dissimilar osteoblastic potential.</a> Stem Cells and Development. 22, 1275-1287 (2013).</li><li>Rivera, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blackwater+fever+like+in+murine+malaria.">Blackwater fever like in murine malaria.</a> Parasitology Research. 112, 1021-1029 (2013).</li><li>Pochsmpally, R., Procko, D. J., Phinney, D. G., Bunnell, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony+Forming+Units+Assays+for+MSCs.">Colony Forming Units Assays for MSCs.</a> Mesenchymal Stem Cells. Methods and Protocols. , (2008).</li></ol>

The authors have nothing to disclose.

The stem cells that were obtained from human amniotic membrane (Figure 1) showed a fibroblast-like morphology, similar to mesenchymal cells (Figure 2) and were principally MSCs. In addition, flow cytometry assays revealed that these cells were positive for mesenchymal cell markers (CD73, CD90, and CD105) and negative for hematopoietic lineage markers (CD34, CD45) (Figure 3). Also, the AM-hMSCs isolated in this work present the capability of forming CFUs (Figure 6). Similarly, the mesenchymal stem cells isolated in this form retained the ability to attach to osteoinductor biomaterial (Figure 4), and proliferate on the scaffold (Figure 5).
For these reasons, the culture method of AM-hMSCs on macroporous bone biomaterial that was used in this work represents an opportunity to insert cells within injured bone tissue and induce its differentiation to the osteoblastic lineage. This culture system does not require the addition of osteoblastic inductors to maintain the osteoblast phenotype and osteoblastic differentiation process because NKB is an osteoconductive andosteoinductive material8. The use of waste tissues, such as human placenta, represents a simple and ethical alternative to access a population of stem cells with the potential to differentiate into MSCs, in comparison with other sources that frequently involve invasive methods or low cell yield.
The proposed culture method could be extrapolated to in vivo systems because the cells are seeded in micro-mass on the top surface material, and the cells colonize and adhere to the entire biomaterial surface after the appropriate incubation times. Furthermore, sophisticated reagents, equipment and procedures are not required, unlike existing methods when implementing this technique are the micro-mass cultureas well as the slow and steady deposition of the cellular aliquot on the material to ensure that the cells preferentially interact with the biomaterial, and not with the bottom of the plate. Another critical point is the use of pre-wetted material before culturing the cells, as this ensures that the cells will have the necessary nutrients from the culture medium when they are integrated into the material regardless if they are on the surface or within the pores. Finally, the use of an osteoinductive biomaterial is critical to this process because it avoids the addition of external factors to induce and maintain osteoblastic differentiation. The limitation of the technique is that it is based on the osteoinductive potential of the bovine matrix; however, the proposed culture system can be extrapolated to any porous material with a pore distribution of 20 to 200 &#181;m. Therefore, the procedure presented in this work is an alternative method for culturing mesenchymal stem cells in various materials that are employed for tissue engineering, especially for bone regeneration.

There pair and regeneration of tissuesis one of themain objectives of medical science because a lesion in a tissue ororgan can be incapacitating and, in some cases, lethal. In context, the health workers and researchers are constantly searching techniques, methods and tools to provide new therapeutic alternativesto promote there pair-regeneration process of damaged tissue. Therefore, biomedical sciences have focused on the study of mesenchymal stem cells (MSCs), especially adult stem cells, because this cell lineage represents an ideal model to study the tissue regeneration process, due to its potential to differentiate to ectodermal, mesodermal, and endodermal cell lineages However, to make therapy with MSCs a reality, the characterization of thetherapeutic potential of MSCs must be accompanied by the implementation of new cell culture techniques using biomaterials and cellular scaffolds that guarantee the desired behavior of the MSCs in the host tissue.
The principal challenge to overcome in the implementation of a culture with MSCs in bone regeneration is to secure cell adhesion on the selected biomaterial, which generally presents a porous structure with adistribution of pores ranging from micrometers to millimeters have been employed but frequently require sophisticated reagents, equipment, and procedures that alter the behavior of cells. These procedures are suitable for in vitro estimation, but not for the extrapolation and implementation in complex open systems, such as human or in vivo assays. Techniques that focus on ensuring cell adhesion to random topographies, such as porous bone materials, have been studied by the adhesion of the material to the bottom of culture wells and employed collagens and agarose polymers. For these methods, the cells that were seeded on the surface were retained in the pores and on the top surface. However, this aspect cannot be ensured in an in vivo system, in which the body fluids surrounding the biomaterial could drag the cells from the desired site on porous scaffolds is the use of ultrasonic stimulation, which requires sophisticated equipment but promotes the expression of osteoblastic markers in in vitro assays5. The use of continuous cultures requires bioreactors, which guarantee a homogeneous distribution of nutrients in the culture medium; however, a significant percentage of the cultured cells is lost when replacing the culture medium due to the flow rate that is used in this technique and the medium adhering to the walls of the equipment. These issues increase the cellular mass that is necessary for this type of culture and the time for obtaining a sufficient number of cells7. Thus, the methodology presented in this work represents a technique that should be extrapolated to in vivo systems because the cells are seeded in micro-mass on a top surface material, and after the appropriate incubation times, 60% of the initial cellular mass is adhered. Moreover, this technique does not require the addition of osteoblastic inductors (e.g., dexamethasone, ascorbic acid, or beta-glycerophosphate) or ultrasonic stimuli to induce osteoblastic differentiation and maintenance of the osteoblast phenotype because NKB is an osteoconductive and osteoinductive scaffolds8, the method for the culture of MSCs from the human amniotic membrane (AM-hMSCs) on macroporous bone biomaterial presented in this work representsapossibility to insert cells into damaged bone tissue and induce differentiation to the osteoblastic lineage.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>sterile phosphate buffered saline</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>10010023</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>10378016</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>26140111</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with high glucose</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>11965118</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>collagenase type II</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>17101015</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>3mM calcium chloride</td>
			<td>SIGMA_ALDRICH</td>
			<td>C5670</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>trypsin/0.5mM EDTA solution</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>R001100</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>15250061</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>phycoerythrin (PerCP)-conjugated CD73</td>
			<td>BD Pharmigen</td>
			<td>562245</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>FITC- CD90,</td>
			<td>BD Pharmigen</td>
			<td>562245</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>PE-CD34</td>
			<td>BD Pharmigen</td>
			<td>562245</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>FITC-CD45</td>
			<td>BD Pharmigen</td>
			<td>562245</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>PE-CD105</td>
			<td>BD Pharmigen</td>
			<td>562245</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>BD FACSCalibur Flow Cytometer</td>
			<td>BD Pharmigen</td>
			<td>BD FACSCalibur</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>alamarBlue (AB)</td>
			<td>LIFE TECHNOLOGIES</td>
			<td>DAL1025</td>
			<td>proliferation cell assay</td>
		</tr>
		<tr>
			<td>SUNRISE-reader</td>
			<td>TECAN, Germany</td>
			<td>Sunrise</td>
			<td>proliferation cell assay</td>
		</tr>
	</tbody>
</table>

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.

Human mesenchymal stem cell isolation
After mechanical separation of the AM from the chorion using blunt dissection (Figure 1), an adherent cell population was obtained by trypsin and collagenase II digestion. These cell populations attached in the culture dish presenta fibroblast-like cell morphology at 3 days post isolation as shown in the optical micrograph (Figure 2A) and scanning electron micrograph (Figure 2B).The placentas used in this work were obtained from healthy donor mothers withprevious informed consent.
Cell characterization by flow cytometry
The cell population that was obtained from the AM was strongly reactive to the surface mesenchymal markers CD90+ (93.5%&#160;&#177;&#160;6.85) and CD73+ and CD105+ (79%&#160;&#177;&#160;3.46) and showed a negative reaction to hematopoietic markers such as CD34- and CD45. Thus, the AM-hMSCs used in this work were mesenchymal, in accordancewith information previously reported by Leyva et al. (Figure 3). Additionally, this result coincides with the immunophenotype characteristics that were established by the International Society for Cellular Therapy (ISCT) for mesenchymal stem cells.
Cell adhesion on bone matrix
The scanning micrographs show the behavior of the AM-hMSCs seven days after layering on the NKB. The surface of the biomaterial was covered with spherical cells (day one), and some spreading cells apparently attached to the bovine matrix surface on the seventh day. At higher magnification, the spreading cells appeared to be in close contact via filipodial processes (FL) (Figure 4).
Cell proliferation on bone matrix
The results of the AB assay showed a small decrease in relative absorbance units (RAU) of bovine matrix condition (+bone matrix) after 1 day of incubation and then on the fifth day, the presence of the scaffold induces an increase in the cell proliferation statistically significant in comparison with the culture performed in the absence of bovine matrix (-bone matrix) (Figure 5).
Colony Forming Unit
The CFU is a traditionally assay of stem cells. The AM-hMSCs isolated in this work preserved its capability to form discrete colonies at 14 days in culture.
<img alt="Figure 1" src="/files/ftp_upload/51999/51999fig1highres.jpg" /> 
Figure 1: Isolation of amniotic membrane. (A) The umbilical cord (UC) is held with one hand to identify the region in which the amniotic membrane is obtained. (B) The amniotic membrane (AM) resembles a translucent film without blood innervations.
<img alt="Figure 2" src="/files/ftp_upload/51999/51999fig2highres.jpg" /> 
Figure 2: Morphological characteristics of stem cells from the human amniotic membrane. AM-hMSC cultures at 3 days post-isolation were observed using an optical microscope and shown the fibroblast-like morphology.
<img alt="Figure 3" src="/files/ftp_upload/51999/51999fig3highres.jpg" /> 
Figure 3: Flow cytometry characterization. The cells from human amniotic membrane were mostly mesenchymal cells because they were positive for mesenchymal markers (CD73, CD90, CD105), as shown by the displacement of histogram to the right, and negative for hematopoietic markers (CD34 and CD45), as confirmed by the displacement of the histogram to the left. The antigens are shown in red histograms, while the control isotypesfor the PE and FITC fluorochromes are observed in black.
<img alt="Figure 4" src="/files/ftp_upload/51999/51999fig4highres.jpg" /> 
Figure 4: Cell adhesion on bovine bone matrix. A series of SEM images showing attachment of the cells to pores of the biomaterial. (A) Panoramic vision of a NKB pore with several cells adhered to the biomaterial in the spherical state (CS) and flattened (CF) on the surface of the biomaterial, is also possible to appreciate the bone lacuna indicates as (Lac) at three day of culture on the bovine matrix. (B) Amplification of the cell in the process of adapting at the surface of the material through filipodial projections. (C) Interaction between two cells adhered and flattened on the bovine matrix (C1, cell 1; and C2, cell 2). <a href="https://www.jove.com/files/ftp_upload/51999/51999fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/51999/51999fig5highres.jpg" /> 
Figure 5: Proliferation of cells on bovine bone matrix. Cell proliferation was evaluated by AB reduction and expressed in relative absorbance units along 7 days of cultivation. Results are shown the influence of bone matrix (+bone matrix) in AM-hMSCs proliferation which was statically different in comparison with the negative condition (-bone matrix). (*p &#60;0.05)
<img alt="Figure 6" src="/files/ftp_upload/51999/51999fig6highres.jpg" /> 
Figure 6: CFU assay of MSCs initially plated at varying densities and incubated for 14 days (mean*/- SD, n = 10).
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>Cells adhered</td>
			<td>Cell adhesion (%)</td>
		</tr>
		<tr>
			<td>Cells on the bottom&#160;</td>
			<td>187667 &#177; 1208</td>
			<td>62.47</td>
		</tr>
		<tr>
			<td>Cells on the scaffold</td>
			<td>312333 &#177;1208</td>
			<td>37.53</td>
		</tr>
	</tbody>
</table>
Table 1: Efficiency of adhesion on bovine matrix. The cells adhered to the bottom of the plate dish and the cells adhering on the scaffold were counted by hemocytometer after being recovered by trypsinization.The data presented in the table correspond to two experiment independent for triplicate &#177; standard deviation

Watch this Scientific Journal Video about Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix at JoVE.com

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 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 ...

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Developmental Biology

27.4K visualizações.  Universidad Nacional Autónoma de México (UNAM). 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.

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

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 ...

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

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 ...

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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

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

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

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

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