Processing procedures of umbilical cord blood (Baba K et al. Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study. J Craniomaxillofac. 2013)
3 Treatment of the UC
The UC has two umbilical arteries and one umbilical vein, and blood is contained in the lumen of the arteries and vein. MSCs are present in the vascular endothelium and UCB as well [24, 30]. When assessing MSCs derived from WJ (WJ-MSCs), it is necessary to deal specimens in order not to allow any contamination of cells that are contained in the vascular wall and blood. WJ-MSCs have higher expansion and engraftment capacities compared to BM-MSCs . On the other hand, the success rate of WJ-MSC isolation was only 63 % from UCB .
We used WJ in the culture as described below and cultured WJ-MSCs by using Dulbecco’s modified Eagle’s medium (DMEM) that are used for the selective culture of MSCs. After the collection of UCB, the UC was washed with phosphate buffered saline up to the moment when no attached blood remained. After the removal of the periumbilical membrane, umbilical arteries, and umbilical vein, WJ was cut into about 5-mm sections. These sections were incubated by using DMEM added with 10 % autoserum derived from UCB in the 25-cm2 flask under the conditions of 5 % CO2 and 37 °C. Media were replaced for the first time at 1 week after the onset of the culture and every 3 days thereafter. Following the emergence of adhesive spindle-shaped cells, the WJ sections were removed at about 2 weeks after the onset of the culture (Fig. 27.2). These cells resembled fibroblasts and were adhesive and spindle-shaped, thus demonstrating no discordance with the morphological features of MSCs .
Pretreatment of the umbilical cord (Baba K et al. Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived autoserum. J Craniomaxillofac. 2012). The umbilical cord was collected after the expulsion of the placenta. (a) The umbilical cord was cut into about 10-cm sections and was washed with phosphate buffered saline up to the moment when no attached blood remained. (b) Cross section of the umbilical cord. The umbilical cord was washed up to the moment when no attached blood remained also in the vascular lumen. The cross section identified the umbilical vein and arteries. (c) After the removal of the periumbilical membrane, umbilical arteries, and umbilical vein, Wharton’s jelly was cut into about 5-mm sections (white arrow). (d) Wharton’s jelly was cultured in Dulbecco’s modified Eagle’s medium added with 10 % autoserum. (e) Outgrowth cells from Wharton’s jelly that were observed at 2 weeks after the onset of the culture. (f) Outgrowth cells were adhesive spindle-shaped cells
4 Features of WJ-MSCs
Stem cells may be categorized to embryonic stem cells (ESCs) and adult stem cells (ASCs), and the latter cells are categorized mainly into hematopoietic stem cells and MSCs . These cells present different features : ESCs are considered to have the potential of differentiating into almost all tissues, while MSCs do not . In 2006, the International Society of Cellular Therapy (ISCT) proposed the minimal criteria for defining MSCs: the cells can adhere to the plastic under standard culture conditions and should have the multipotency to differentiate into osteoblasts, adipocytes, and chondrocytes in an in vitro study as demonstrated by specific staining .
The general features of MSCs are (1) self-renewal capability and (2) plurilineage or multilineage differentiation potential [32–34]. We verified the self-renewal capability of WJ-MSCs based on the cell growth curve. Furthermore, we confirmed WJ-MSCs’ multilineage differentiation potential into bone and fat. WJ-MSCs, which had been induced to differentiation into osteoblasts, were stained extracellularly and diffusely with alizarin red, verifying the presence of calcium. WJ-MSCs, which had been induced to differentiation into adipose cells, showed intracellular lipid droplets that were stained with oil red (Fig. 27.3). WJ-MSCs differentiate into cartilage [35, 36], nerve [35, 36], myocardium , insulin-producing cell , hepatocyte , and other tissues. WJ-MSCs possess flexible differentiation potential [32–35].
In vitro assessment of the osteoblastic and adipogenic differentiation potential of Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs) (Baba K et al. Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived autoserum. J Craniomaxillofac. 2012) WJ-MSCs were induced to differentiate into osteoblasts and adipose cells. (a) Staining with alizarin red. The specimens of WJ-MSCs, which had been induced to differentiation into osteoblasts, were stained extracellularly and diffusely with alizarin red. (b) Staining with oil red. The specimens of WJ-MSCs, which had been induced to differentiation into adipose cells, showed intracellular lipid droplets that were stained with oil red (white arrows)
In recent years, the elucidation of cell surface markers (CSMs) has advanced. According to the ISCT’s proposal, MSCs positively express CD73 (SH2), CD90, and CD105 (SH3) and negatively express CD34, CD45, CD14/CD11b, CD79α, CD19, and HLA-DR . CSMs of different tissue origins are expressed differently in MSCs. CD73, CD90, CD105, and other markers are expressed in WJ-MSCs . WJ-MSCs express the typical MSCs markers (CD105, CD73, and CD90) and but do not express CD34, CD45, CD14, CD19, and HLA-DR . Furthermore, WJ-MSCs express a trace amount of CSMs that are common to ESCs (e.g., Oct4, NANOG, DNMT3B, and GABRB3) [26, 40]. Culture has been suggested to possibly modify the expression of CSMs , and this modification may influence the results from previous studies.
The specification of CSMs, which are specific to WJ-MSCs with excellent osteogenic potential, in the future will allow the elective collection of the relevant cells and will permit promise for more efficient and stable osteogenesis.
5 Osteogenic Potential of WJ-MSCs Using UCB-Derived Fibrin Net (UCB-FN)
MSCs are featured, depending on their origins, to generate tissues that are prone or less prone to differentiation . Cells to be used as osteogenic cells in clinical settings are required to have sufficient potential to differentiate into osteoblasts. WJ-MSCs are believed to have features shared by ESCs and adult MSCs and have been reported to possess flexible differentiation potential . The extent of “differentiation potential flexibility”—a characteristic of WJ-MCSs—may require more restrictive conditions for the induction of differentiation into target tissue. Will WJ-MSCs differentiate into osteoblasts if arranging conditions for culture? A number of authors have reported the osteogenic potential of WJ-MSCs in in vitro studies on cell activity, development of osteoblast marker expression, CMSs, and other study topics [22, 26, 35, 40]. We examined the osteogenic potential of WJ-MSCs in an in vivo study, in which WJ-MSCs were used as osteogenic cells, UCB-derived fibrin (UCB-fibrin) as scaffold, UCB-PRP as growth factor, and UCB-derived serum (UCB-autoserum) as serum for culture for future clinical application. This study was designed to provide all three elements required for regenerative medicine and autoserum for culture by means of autologous tissues. The availability of autologous tissues as biomedical materials for regenerative medicine suggests that these may be safe to use in pediatric patients.
5.1 Assessment of UCB-FN
Fibrin is a physiological material that is produced in tissue damage (e.g., bone fracture) and is very compatible with surrounding tissues and cells. The structures of thawed UCB-fibrin resembled those of peripheral blood from adults in which fibrin components were mixed homogeneously.
The structures of thawed UCB-fibrin were examined by scanning electron microscopy (SEM). UCB-fibrin seeded with WJ-MSCs was also examined by SEM, which revealed the reticulated structures of thawed fibrin in which fibrin fibers were intertangled. The structures were preserved even after cryopreservation and thawing. These three-dimensional structures were relatively homogeneous in both the superficial and deep layers (Fig. 27.4). In recent years, the differentiation of MSCs has been indicated to require three-dimensional cultures [42–45]. SEM verified that the fibrin structures preserved their three-dimensionality to deep layers without any artificial manipulation, leading us to consider that the structures are compatible with the culture of MSCs.
Umbilical cord blood-derived fibrin (UCB-fibrin) immediately after thawing (Baba K et al. Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study. J Craniomaxillofac. 2013). (a) Macroscopic appearance. (b) Scanning electron microscopy image (SEM) of the UCB-fibrin surface showing reticular structures. (c) SEM image of the UCB-fibrin cross section showing reticular structures that are preserved to deep layers
Furthermore, autologous WJ-MSCs naturally infiltrated into UCB-fibrin only by seeding. This result drives us to consider that UCB-fibrin has good compatibility with autologous cells. Although not investigated in detail, one of permissible reasons is that there are some unelucidated actions of several proteins [46–48] that were contained in serum and other fluids present in the surface of or spaces between fibrin fibers. UCB-fibrin was simple to prepare and was easy to handle in the entire process of manipulation. Fitting to the configuration of the implantation site was possible by superimposing multiple sections, indicating the readily adaptable feature of UCB-fibrin to the defect morphology when applied in clinical settings.
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5.2 Osteogenic Potential of WJ-MSCs In Vivo
UCB-fibrin, which was cut into pieces of 5 mm in size, was put into the flask, and primary outgrowth cells were seeded (1 × 105 cells/piece). DMEM was used to conduct 1-week culture to wait for cell infiltration into UCB-fibrin. Afterwards, the medium was changed to NH OsteoDiff Medium, and this medium was used for differentiation induction to osteoblasts during 3 weeks. When the medium was changed to NH OsteoDiff Medium, UCB-PRP was added to the medium at the ratio of 300 nL/mL only at the first time. UCB-PRP and specimens were implanted subcutaneously into the dorsum of seven male nude mice BALB/Ca Jcl-nu nu/nu aged 5 weeks.
Specimens were removed 6 weeks later. In order to remove specimens, mice were subjected to euthanasia in accordance with our institution’s guidelines on methods to kill and dispose of animals. At the time of removal, specimens appeared yellowish white, had slightly elastic rigidity, and showed several capillary vessels on the surface (Fig. 27.5a, b). These removed specimens were assessed as described below.
Specimens obtained when combining Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) with umbilical cord blood-derived fibrin (UCB-fibrin) in the in vivo study (Baba K et al. Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study. J Craniomaxillofac. 2013). (a) Macroscopic appearance of specimens found subcutaneously in the mouse dorsum at the time of removal. The specimens showed capillary vessels on their surface. (b) The removed specimens had a yellowish white appearance and were hardened at the time of removal. (c) Image of micro-computed tomography showing high-density areas
Micro-computed tomography (microCT)
MicroCT revealed internal radiopaque areas of the specimens that were suggestive of calcification (Fig. 27.5c).
Hematoxylin and eosin (HE) staining and alizarin red staining
The HE-stained specimens included areas that were well stained with hematoxylin which were suggestive of ectopic calcification (Fig. 27.6a). The same areas were positive for alizarin red staining and exhibited ectopic calcium (Fig. 27.6b). Cells with a definite nucleolus were found adjacent to these areas. Several sites of ectopic calcification included a scanty number of cells internally (Fig. 27.6a). Specimens exhibited extremely small eosin-stained parts but included no cells, providing no histological picture suggestive of definite bone tissue.
Specimens obtained when combining Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) with umbilical cord blood-derived fibrin (UCB-fibrin) in the in vivo study (Baba K et al. Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study. J Craniomaxillofac. 2013). (a) Image of a specimen stained with hematoxylin and eosin (HE staining), showing areas of ectopic calcification in its interior. (b) Image of the specimen shown in Panel a, which was stained with alizarin red, exhibiting positivity in the areas corresponding to ectopic calcification in the image of HE staining. (c) Image of the specimen shown in Panel a, which was immunohistochemically stained with the antimitochondrial antibody, exhibiting positive cells in the periphery of the site of ectopic calcification
Immunohistochemical staining of human mitochondria
Cells positive for antihuman mitochondria antibody staining were located in the periphery of ectopic calcification within the fibrin (Fig. 27.6c).
SEM with energy dispersive X-ray spectrometry (SEM-EDX) and transmission electron microscopy (TEM)
The surface of the specimens was covered with cells arrayed in a squamous manner (Fig. 27.7a). A specimen exhibited numbers of spindle-shaped cells and granular materials (Fig. 27.7b). Granular materials were accumulated in some areas and were continuous with cell processes (Fig. 27.7c). We speculate that these granular materials had been secreted from cells. SEM-EDX of these granular materials revealed calcium and phosphorus, with a Ca/P molar ratio of 1.67 (Fig. 27.7d). We found that the materials have a Ca/P molar ratio close to that of hydroxyapatite. The formation of secretory vesicles was observed in cells by TEM (Fig. 27.8). Analyzed together, we interpret that cells that differentiated from human-derived cells, i.e., WJ-MSCs, caused the accumulation of hydroxyapatite.
Images of scanning electron microscopy of specimens when combining Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) and umbilical cord blood-derived fibrin (UCB-fibrin) in an in vivo study (Baba K et al. Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study. J Craniomaxillofac. 2013). (a) The surface of a specimen covered with cells that are arrayed in a squamous manner. (b) Cross section of a specimen showing spindle-shaped cells and granular materials in its interior. (c) Magnified image of the cross section showing the continuation of cell processes and granular materials. (d) Analytical electron microgram of granular material showing a Ca/P molar ratio of 1.67. (e) The analyzed site is indicated with a white arrow
Formation of secretory vesicles (white arrows) was observed in cells by transmission electron microscopy (Baba K et al. Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study. J Craniomaxillofac. 2013)
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