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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 39  |  Issue : 1  |  Page : 6-12

Inducing hepatogenic differentiation of human mesenchymal stem cells derived from umbilical cord blood


1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
3 Department of Gynecology and Obstetrics, Faculty of Medicine, Tanta University, Tanta, Egypt
4 Ministry of Health Hospitals, Tanta, Egypt

Date of Submission24-Aug-2013
Date of Acceptance28-Oct-2013
Date of Web Publication29-Jan-2014

Correspondence Address:
Dareen A Mohamed
Department of Pathology, Faculty of Medicine, Tanta University, Tanta, 31111
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.124838

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  Abstract 

Background and aim The present work aimed to study the ability of human mesenchymal stem cells (MSCs) derived from umbilical cord blood (UCB) to transdifferentiate into hepatocytes and to assess the characterization of the transformed cells in vitro.
Materials and methods MSCs were isolated from 30 UCB samples collected aseptically from completely separated placentas of full-term deliveries. The separation of MSCs was carried out from freshly isolated mononuclear cells suspensions in a primary culture for 2 weeks. MSCs were identified before induction by cytochemical stain for periodic acid-Schiff and morphology. Then, they were induced to transdifferentiate into hepatocytes by hepatogenic medium, and differentiation of hepatocytes was confirmed by morphological and functional assessments of urea production, glycogen storage, and immunocytochemistry of α-fetoprotein.
Results We successfully isolated 12 MSCs units from 30 full-term UCB samples (40%). The cells showed positive staining for periodic acid-Schiff, indicating that they retain the characteristics of MSCs. The response of UCB-derived MSCs to hepatogenic medium containing hepatocyte growth factor was assessed by changes in the morphology that occurred within 2 weeks in most of the cells. The morphological changes were observed closely and the induction process was monitored by immunocytochemistry for α-fetoprotein, urea production, and glycogen storage. The results were positive in most of the induced hepatocytes, with some variations from sample to sample because of the variability in the cell count in each UCB unit used.
Conclusion We concluded that MSCs in human UCB can differentiate into viable functioning hepatocytes when cultured in hepatogenic conditioned medium in vitro.

Keywords: stem cells, hepatogenic


How to cite this article:
Elzamarany EA, Nosair NA, Mohamed DA, Moustafa MZ, Balah GM. Inducing hepatogenic differentiation of human mesenchymal stem cells derived from umbilical cord blood. Egypt J Haematol 2014;39:6-12

How to cite this URL:
Elzamarany EA, Nosair NA, Mohamed DA, Moustafa MZ, Balah GM. Inducing hepatogenic differentiation of human mesenchymal stem cells derived from umbilical cord blood. Egypt J Haematol [serial online] 2014 [cited 2017 Aug 16];39:6-12. Available from: http://www.ehj.eg.net/text.asp?2014/39/1/6/124838


  Introduction Top


Every year, many individuals worldwide die from liver diseases [1] . The liver has a well-documented ability to regenerate itself following injury or partial resection. It has long been known that hepatocytes are the major contributors to liver regeneration [2] . However, when liver damage is very severe or when carcinogens or toxins inhibit expansion of hepatocytes, regenerative capacity fails and then fibrosis and eventually cirrhosis develops [3] .

The only curative treatment for advanced liver cirrhosis is orthotropic liver transplantation (OLT), which is an effective treatment for end-stage cirrhosis and many liver-based inherited metabolic disorders. However, OLT has several limitations such as a long waiting list, high cost, and several major complications [1] .

Cell transplantation is less invasive than whole-organ transplantation. It can be performed repeatedly, and has been used to bridge patients to whole-organ transplantation [4] . Cell transplantation can decrease mortality in acute liver failure and can be used for the treatment of metabolic liver diseases [5] . Hepatocyte transplantation is an alternative for OLT in the treatment of end-stage cirrhosis and liver-based inherited metabolic disorders [6] . Despite positive reports, the application of hepatocyte transplantation in humans is limited [7] . The reason for this discrepancy is the success of OLT and limited availability of human hepatocytes. Thus, there is a need to find an easily available cell type equivalent to primary hepatocytes [8] .

Mesenchymal stem cells (MSCs) are self-renewing cells that can give rise to various mesodermally derived tissues [9] . Because of readily available sources and expansion potential exceeding one billion-fold in culture, MSCs represent an essential ingredient required for successful bioengineering of human tissue [10] . MSCs are plastic-adherent stem cells expressing CD73, CD90, and CD105, lacking MHC class II and lymphoid markers such as CD34 and CD45, and showing the capacity to differentiate into a variety of cell types [9] .

Originally isolated from the bone marrow, cells with properties similar to those described for MSC have been derived from a number of other tissues, including periosteum, synovium, lung, adipose tissue, skeletal muscle, umbilical cord, and cord blood [11] .

Human umbilical cord blood (UCB) is a readily available, noninvasive source of hematopoietic stem cells and these attributes would also make it attractive for producing lines of MSCs for clinical applications [12] .

Although MSCs represent a promising source, technical issues in terms of isolation from a given tissue, expansion, differentiation into hepatocyte-like cells procedure, and, especially, making this process an economically viable alternative still remains a challenge [13] .

The aim of the present work is to study the ability of human MSCs derived from UCB to transdifferentiate into hepatocytes and to assess the characterization of the transformed cells in vitro.


  Materials and methods Top


Ethical committee approval was obtained for the study from the Ethical Committee of Faculty of Medicine, Tanta University. Informed consent was received from the mothers for collection of 30 full-term human UCB samples at delivery in the Department of Gynecology and Obstetrics at Tanta University Hospital.

Reagents

The following reagents were used: calcium-free and magnesium-free PBS and fetal bovine serum (FBS) (Invitrogen, Carlsbad, California, USA), Ficoll-hypaque solution (density, 1.077 g/l), fungizone and HEPES medium (Bioscience, Surrey, UK), sterile cell culture RPMI with l-glutamine (Lonza, Walkersville, Maryland, USA), hepatocyte growth factor (HGF) (R&D System, Wiesbaden, Germany), and penicillin/streptomycin and trypsin 0.25% (1×) with 0.1 EDTA (Sigma, Gillingham, UK).

Collection and storage of umbilical cord blood

Using established protocols [14] , immediately after delivery, the umbilical cord was double-clamped and dissected. Then, after removal of the baby and separation of the placenta, UCB was harvested aseptically by umbilical vein puncture using a 20 ml sterile syringe. After vein puncture, 20 ml of UCB was collected using gentle suction and emptied into a sterile Falcon tube (50 ml) that contained 4 ml citrate phosphate dextrose as an anticoagulant, followed by inversion several times and labeling of the sample. Samples were transported to the main laboratories of Tanta University Hospitals in less than 12 h; during this time, they were preserved at room temperature (22 ± 4°C).

Separation of human umbilical cord blood mononuclear cells

Human UCB mononuclear cells (MNCs) were separated from UCB by density gradient centrifugation [15] . Under complete aseptic conditions, the fresh cord blood was diluted with sterile calcium-free and magnesium-free PBS in the ratio of 1 : 2 and they were mixed well. Gently, the diluted blood was layered onto the top of warm Ficoll-hypaque density. After centrifugation, the MNCs fraction was collected into a sterile conical centrifuge tube. The cells were washed twice in 50 ml cold PBS. Cell pellet was counted using a hemocytometer. Cell counting and viability were assessed using the vital stain trypan blue (0.04%) exclusion dye (Sigma).

Isolation and culture of mesenchymal stem cells

The MNC suspensions were seeded at concentrations of 1 × 10 6 cells/cm 2 and allowed to adhere to 25 cm 2 tissue culture plastic flasks (Corning, USA), incubated in 7 ml of the fresh complete nutrient medium, which included the following: RPMI with 1% l-glutamine, 10% heat-inactivated FBS, 1% penicillin/streptomycin, and 0.5% fungizone. For proper adherence of the cells, the flasks were incubated in a horizontal position in a humidified incubator at 37°C and 5% CO 2 and media pH 7.2-7.3. The media were examined daily both visually and by an inverted phase-contrast microscope for assessment of cell condition, morphology, and signs of microbial contamination. On day 7, the media were replaced and on day 14, cells were examined microscopically to ensure 50-70% confluence, normal cell morphology, and lake of bacterial contamination. Then, these cells were harvested by trypsinization [16] .

Identification of mesenchymal stem cells

MSCs were identified morphologically and using periodic acid-Schiff (PAS) stain. Pink cytoplasmic staining was considered positive and blue color was considered negative.

Hepatogenic differentiation of mesenchymal stem cells

MSCs that were harvested from the primary culture bottle with trypsin were recultured in a tissue culture plastic flask (25 cm 2 ) for 3-4 weeks in hepatogenic medium composed of the following: 2 ml RPMI with l-glutamine, 1 ml HEPES medium, 20% FBS, 100 μl penicillin/streptomycin, 0.5-1% fungizone, and 20 ng/ml HGF [17] . The media were examined daily and changed every 4 days and the changed media were kept at −20°C for urea assay. On day 20, samples were harvested by trypsinization for the detection of α-fetoprotein (AFP) by immuncytochemical stain and the changed media were stored at −20°C for urea assay. On day 28, cells were examined microscopically to ensure 50-70% confluence, normal cell morphology, and lake of bacterial contamination. Then, these cells were harvested by trypsinization.

Identification of hepatocyte formation was carried out by the following

  1. Morphologically: Microscopic examination of morphological changes.
  2. Cytologic smear: Cells were harvested from cultures and cytospins were prepared by plating cells on glass slides. After 24 h of culture in an incubator, cytospins were fixed with dehydrated alcohol/dimethylketone at 1 : 1 for 20 min and immunocytochemistry (ICC) analysis was carried out. Slides were counterstained with hematoxylin.
  3. Functionally: Detection of urea using the colorimetric method (modified urease-Berthelot method) on days 4, 8, 12, 16, 20, 24, and 28, detection of glycogen production by PAS stain on days 20, 24, and 28, and also immunocytochemical stain to demonstrate AFP production.


Periodic acid-Schiff for glycogen

Slides were oxidized in 1% periodic acid (Sigma) for 5 min and rinsed three times in distal H 2 O. Afterward, slides were treated with Schiff's reagent (Sigma) for 15 min, rinsed in deionized H 2 O for 5-10 min, stained with Mayer's hematoxylin for 1 min, and rinsed in deionized H 2 O. Glycogen storage was observed as accumulation of magenta staining.

Detection of α-fetoprotein by immunocytochemistry

Cytologic smears were soaked in antigen retrieval fluid (DakoCytomation; Dako, USA) for microwave antigen retrieval for 15 min; then, they were ready for the application of the primary antibody (mouse monoclonal anti-mouse AFP). Three drops of AFP antibody were added to each sample. Smears were then incubated in moist chambers overnight, followed by rinsing twice in PBS at pH 7.6. Then, two drops of prediluted biotinylated secondary antibody were added for 20 min at room temperature, followed by rinsing twice in PBS at pH 7.6. Two drops of streptavidin-peroxidase label were added to each smear, followed by incubation for 10 min at room temperature, and then rinsing twice with PBS at pH 7.6. The substrate-chromogen (DAB) mixture (Biogenix) was prepared immediately before use; two drops were added to each smear, followed by incubation for 15 min, with subsequent rinsing in distilled water. Counterstaining with Harris hematoxylin was then performed. Brown granular cytoplasmic staining was considered positive for AFP [18] .

Statistical analysis

Statistical presentation and analysis of the present study were carried out using the mean, SD, and paired t-test by SPSS (version 18; SPSS Inc., Chicago, Illinois, USA). P-value less than 0.05 was considered statistically significant and P-value less than 0.001 was considered highly significant.


  Results Top


The MNCs that were obtained from the fresh UCB samples after Ficoll-hypaque density gradient separation were counted and the viability was assessed using the trypan blue exclusion dye. The cell count ranged from 1.5 to 5.0 × 10 7 cells/ml according to the cord blood volume used and the viability of the MNCs was 95-98%.

Establishment of the primary culture

In this study, we successfully isolated 12 MSCs units from 30 full-term UCB samples (40%). Attached cells were observed at 5-7 days after the initial plating in RPMI with l-glutamine containing 10% FBS and antibiotics.

Characteristics of adherent cells of mesenchymal stem cells culture

Inverted microscopic examination of cells cultured for 7 days showed a discrete adherent cell layer with a heterogeneous morphology (ovoid and epithelioid cells) at the base of the flasks [Figure 1]. After 2 weeks of culture, the UCB-derived MSCs were recognizable as adherent cells with a fibroblast-like appearance as the proportion of ovoid cells reduced and epithelioid cells proliferated and acquired a more defined MSCs morphology. MSCs displayed an elongated, flat fibroblast-like morphology with granules on the surfaces, and when they reached 70% confluence forming spherical cellular masses of different sizes [Figure 2].
Figure 1: Inverted microscopic image of adherent mesenchymal stem cells after 7 days.

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Figure 2: Inverted microscopic image of mesenchymal stem cells after 2 weeks.

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Cytochemical analysis of the cultured cells showed that the mesenchymal-like cells were positive for PAS [Figure 3].
Figure 3: Microscopic image of periodic acid-Schiff stain of mesenchymal stem cells after 2 weeks. Positive staining (right) and negative control counterstained with hematoxylin (left).

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Characterization of the transformed hepatocytes

UCB-derived MSCs had an ability to differentiate into hepatocytes when they were incubated in a hepatogenic medium (containing RPMI with l-glutamine, FBS, penicillin/streptomycin, HEPES medium, and HGF) and maintained for up to 28 days. The cells showed remarkable transition from a bipolar fibroblast-like morphology to a round or an oval shape [Figure 4]. Immediately after exposure to medium containing HGF, the cells began to lose their sharp edges and progressively shrunk, resulting in complete loss of the fibroblastic bipolar morphology. These morphological changes were detected as early as the third day in the region of high cell density, and propagated into the area of low cell density, so that ~70% of the treated cells became oval and round in shape by the end of the second week. The cytoplasmic contraction progressed further during the next 2 weeks, and most of the incubated cells transformed into small round cells that were very similar to oval cells in appearance [Figure 4].
Figure 4: Inverted microscopic image of the transformed hepatocytes.

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Urea production assessment

Urea production and secretion by hepatocytes were detected at various time points throughout differentiation by measuring the urea concentration in the culture media supernatant. Urea produced by the transformed hepatocytes was first detected at a mean concentration of 4.72 ± 1.02 mg/dl on day 20 and increased progressively to 10.28 ± 1.61 mg/dl on day 28, with a statistically significant increase in urea production by the induced hepatocytes with progression of culture [Table 1].
Table 1: Significant increase in the concentration of urea in culture media with time

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Periodic acid-Schiff treatment for glycogen

We analyzed the levels of glycogen storage by PAS staining of the induced hepatocytes on days 20, 24, and 28. Glycogen storage was first observed on day 24, positively stained for PAS. Glycogen storage was observed as accumulation of magenta staining [Figure 5].
Figure 5: Cells stained by periodic acid-Schiff. Glycogen storage was observed as accumulation of magenta.

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Immunocytochemistry of α-fetoprotein

On the 20 th day, the transformed cells were analyzed by ICC. The transformed hepatocytes showed strong positive cytoplasmic expression of the AFP, whereas the noninduced cells were negative [Figure 6].
Figure 6: Microscopic image of immunocytochemistry of α-fetoprotein for induced hepatocytes. Positive cytoplasmic staining of transformed cells (right). Negative staining of noninduced cells (left).

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  Discussion Top


Stem cell transplantation is of great interest as an alternate and promising therapeutic approach. At this point, however, there is still much to be clarified and some challenges remain [19] . Among the potential candidates, umbilical cord-derived stem cells are of particular interest owing to greater proliferation potential and low immunoreactivity [20] .

In order to study the ability of human MSCs derived from UCB to transdifferentiate into hepatocytes, we have separated, characterized, cultured, and propagated a population of UCB-derived MSCs from 30 full-term deliveries.

MSCs separation was found to be critically dependent on culture of freshly isolated MNC suspensions undisturbed for 7 days in RPMI media with 10% FBS and antibiotics. During this period, the cells became adherent to the surface of the flask; they were re-fed and incubated for another week. The separated cells showed MSC morphology and stained positive for PAS.

Our protocol of work was more or less similar to that established by Hong et al. [21] and Kang et al. [22] , who have concluded that UCB is a rich source of MSCs. We successfully isolated 12 MSCs units from 30 full-term UCB samples (40%). Similar to our results, Denner et al. [23] and Kazemnejad et al. [24] succeeded in isolating MSCs from 38.09 and 36.4% of UCB samples, respectively. However, Weiss et al. [25] successfully isolated 11 MSCs units out of 14 UCB samples (78%), whereas Kang et al. [22] isolated only 10 MSCs units from 24 UCB samples (20.4%).

In our study, we found that a high ratio of MSCs among the attached cells in the culture flask was obtained from much fresher UCB samples, and this suggests that this factor is very important; this observation was also supported by a previous study [26] . We also found that change of the media after 24 h of the primary culture led to interruption of the cultured cells and loss of many stem cells because they need a long time to become adherent to the surface of the flask, and this may explain the variations in the success rates between different studies.

In order to verify the nature of the adherent cells at the end of the primary culture, we examined the adherent cells obtained using an inverted phase microscope to confirm the presence of the characteristic morphology of MSCs, and we used cytochemical analysis showing that the mesenchymal-like cells were positive for PAS. PAS stain provided an easy, cheap, and readily available method for verification of MSCs and this finding was previously reported by Rosada et al. [27] .

Many different growth factors involved in the transformation of stem cells into hepatocytes, including acidic fibroblast growth factor (FGF), basic FGF, FGF-4, HGF, and FGF-7, were considered to be important for differentiation of hepatocytes during embryological development. However, only FGF-4 and HGF were considered to promote differentiation of hepatocytes in vitro [28] .

HGF was first identified as a blood-derived mitogen for hepatocytes; it regulates cell growth, motility, and morphogenesis by activating a tyrosine kinase signaling cascade after binding to the proto-oncogenic (c-Met) receptor. HGF and its receptor are the key factors for liver growth and function [29] .

In our study, we used basic HGF in the process of in-vitro transdifferentiation of UCB-derived MSCs to hepatocytes. Our protocol of work depends on cultivation of MSCs in a hepatogenic medium containing RPMI with l-glutamine, HEPES medium, FBS, antibiotics, and HGF for 4 weeks. After exposure to initiation medium, the cells began to lose their sharp edges and progressively shrunk, resulting in complete loss of the fibroblastic bipolar morphology and acquiring the hepatocyte-like one. These results were in agreement with previous studies [21],[30] .

In order to verify the nature of the adherent cells at the end of the hepatogenic culture, we used morphological and functional analysis of hepatocytes characteristics. We showed that morphologically MSCs transformed into small round cells. The diameter of the cells was about 12-16 μm. On day 28, ~70% of the cells were small, round, and epithelioid. These results were more or less similar to those obtained in a study carried out by Kang et al. [22] .

AFP) is a glycoprotein with a molecular mass of 70 kDa. It consists of a single polypeptide chain and ~5% carbohydrates. It is a serum protein that makes up a major portion of the secretory proteins synthesized by mammalian fetal-liver cells during embryonic development, liver regeneration, and hepatocarcinogenesis [31] . In the present work, most of the adherent cells stained positive for AFP by ICC on the 20 th day after initiation of hepatogenic culture; this was also reported by other two studies [32],[33] .

The functional assays for hepatocytes are mainly urea and albumin synthesis; the synthesis of urea is performed by hepatocytes and should be useful as a test for hepatocytes function [34] . In our study, urea production and secretion by hepatocytes were detected at various time points throughout the differentiation process, first appearing at day 20 and increasing till day 28, with a statistically significant increase in urea production by hepatocytes with time. This supports the results obtained by Kang et al. [22] and Ayatollahi et al. [35] . Also, Wang et al. [36] found that urea was not detected on day 6 but a low level was then detected on day 12. Afterwards, urea production increased gradually until day 22 of induction.

Glycogen is a molecule that serves as the secondary long-term energy storage in animal cells, with the primary energy stores being held in adipose tissue. Glycogen is produced primarily by the liver and the muscles [37] . Hepatocytes can generate and store glycogens. In our study, glycogen storage was observed on day 24, being positively stained for PAS, whereas in a study carried out by Ayatollahi et al. [35] , upon treatment with hepatogenic media, glycogen storage was first observed after 21 days of culture. In this respect, differentiated cells in our study acquired hepatocyte functions, including urea secretion and glycogen storage, after 4 weeks of hepatogenic culture.

Although future uses of UCB stem cells as a therapeutic intervention for a diverse group of diseases are still an intriguing possibility, the results of the present study showed that cord blood is a rich source of mesenchymal progenitors. Also, it was considered that the time from sampling to primary culture, the proper technique, and the maintenance of proper culture conditions may be important factors for collection of MSCs on RBMI media.

On the basis of their large ex-vivo expansion capacity, as well as their differentiation potential when cultured on proper hepatogenic media containing HGF, cord blood-derived MSCs may be an attractive cell source for hepatocytes that could be used to resolve many health problems related to advanced or hopeless liver diseases.

From the present study, it can be concluded that UCB represents a safe and accessible source of MSCs, which have an in-vitro hepatogenic potential, resulting in enormous interest in the use of this differentiation potential in the development of cellular medicine for degenerative and metabolic liver diseases.


  Acknowledgements Top


 
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