INTRODUCTION:

Stem cells are having the capacity to revolutionize the forthcoming era of medicine. Basically, stem cells share three criteria:

1. Stem cells show the property of self-renewal having the capacity to renew itself throughout the life and produce exactly the same copy of pre-existing cells. By this process, they maintain their stem population.

2. Stem cells show the property of differentiation to give specialized types of cells like nerve cells, blood cells, muscle cells,etc.

3. Stem cells also have the capacity of renewing the tissues that they can populate.^1,2

These cells play an important role in the developmental process of the embryos and also maintain the adult cells and tissues. Stem cells reside in the body at specific ‘niche’ microenvironments thatare very important for the maintenance of their growth and differentiation. It includes support cells and extracellular matrices. These cells act as repair systems for the body and have the capacity of replacing the other cells by undergoing divisions as throughout the life of the person. Stem cells are blank cells without any specific properties, but under specific conditions, they start to differentiate and acquire specific functions. Stem cells are used for the treatment of various diseases like Alzheimer’s disease, cancer, Parkinson’s disease,etc.^3,4 Stem cells act as the best therapy for genetic and hematological disorders. Human cord blood cells act as an alternative source of stem cells. Umbilical cord consists of hematopoietic stem cells (HSC), mesenchymal stem cells (MSC),andnon-hematopoieticmultipotent stem cells.⁵ However, not much work has been done on the non- hematopoietic multipotent stem cells. Stem cells from theumbilical cord have regenerative properties and can be used for clinical applications.⁶

UMBILICAL CORD STEM CELLS:

These cells are obtained from the postpartum placenta after the delivery of a baby. The remaining blood is collected from the umbilical cord and from the placenta after it gets separated from the newborn. Cord stem cells are those stem cells which are collected during the postnatal period from the umbilical cord and are the source of multipotent stem cells that are capable of forming different types of cells². Cord blood stem cells can also be stored in a cord blood bank and can be used when it is needed by the donor. The umbilical cord is the connecting link between the mother and the developing fetus. It grows from the yolk sac and allantois. It contains a jelly-like substance calledWarton’s Jelly present inside the stromal cells of the umbilical cord.^4,7,8The umbilical cord has two arteries and one vein thatare coiled in a helical pattern. It is surrounded by two arteries that make the cord stiffer and tougher to break.⁹

In 1974, it was declared that the umbilical cord is an origin for the hematopoietic and progenitor stem cells.¹⁰Thecord carries deoxygenated blood to the placenta and takes oxygenated blood from the placenta to the developing fetus. Cord blood cells have an advantage over bone marrow cells. They do not require pure human leukocyte antigen (HLA) tissue matching and having the lesser chances of rejection.¹¹MSC have the ability to form multiple types of cells and are also found to have immunomodulatory properties.^9,12-14 They overcome the limitations of bone marrow-derivedmesenchymal cells as the latter’s differential potential isinversely dependent on age.^9,14,15 In this review, the types of stem cells collected from umbilical cord cell and also its isolation, identification, proliferation and differentiation process will be elaborated. The therapeutic future of the umbilical cord stem cells will also be discussed.

ISOLATION OF CORD BLOOD CELLS:

Cord blood cells are collected at the time of delivery either by in vitro or in vivo methods using specialized apparatus. Cord cells are completely cleaned and kept at a distance from the perineum to prevent their contamination by the maternal blood. A large bore needle is inserted into the umbilical vein thatis linkedto a bag with citrate phosphate dextrose that acts as an anticoagulant. They are preserved in a nitrogen freezer until the transformation. The most commonly used cryopreservantis DMSO. It prevents the cells from getting damaged. Thereafter, 5% dextran and water are added. They are then frozen at the temperature of -80ºC.¹⁶ In some of the cases, they are also stored at -190ºC in the vapor phase inside a liquid nitrogen tank.

Isolation of primary cells from Warton’s jelly consists of 3 steps. In the first step, all the epithelial, vascular and perivascular tissues are removed. Then collagenases, trypsin, proteinase, and hyaluronidases are used for the mechanical grinding and enzymatic digestion of the cells. Thecells are then transformed to the growth containing culture media. Apart from this technique, explants culture method can also be used.¹⁷

ISOLATION OF HSC FROM UMBILICAL CORD:

Isolation is based on the expressing surface marker antigens and also on the lack of the expressing lineage-specific antigens. CD34, CD133 andthe immune-magnetic selection process is used for this. A positive selection process is done by using specific monoclonal antibodies. These antibodies are conjugated with paramagnetic microbeads.¹⁸Negative selection of lineage negative cells show that the unwanted cells are found to be labeled against known markers. These are for the mature hematopoietic cells that are retained in the column.¹⁹The cells that do nottake the label just pass by the column and are taken as Lin- fraction.¹⁹ The most subtle and fast method to isolate hematopoietic cells is immunogenic cell sorting system MACS.²⁰

ISOLATION OF MSC FROM UMBILICAL CORD:

MSC have till been now isolated from many of the cell types including bone marrow, dental pulp and liver to name a few. For a long time, these stem cells were being isolated from the bone marrow but this method has many demerits.^9,20 So, the isolation of MSC from umbilical Cord is now considered to be a better option. The method is easier and without any ethical concern. It is also found to overcome the limitations of the previous method.

Both the blood and the tissue of the umbilical cord are found to be the source of the MSC.^9,20However, the umbilical cord tissue is used mostly for isolation of the MSC instead of umbilical cord blood because of the variety of reasons that includes the low number of MSC obtained after the isolation and also the unavailability of a proper technique for the same.^9,20,21

The method of isolation of these stem cells varies from scientist to scientist depending on the facility and the need.^9,14 Some of them use the method in which the MSC are isolated from the umbilical cord vessels by scarping off the Wharton’s jelly and then giving an enzymatic treatment to the tissue ^23,24. Others have used methods in which the umbilical cord is cut down to smaller parts and the vessel is stripped followed by the direct enzymatic treatment for the remaining umbilical cord.^17,21,22Some groups have also adopted methods which avoid enzymatic treatment and includes explants of the cord for setting up the culture.²⁴ This method is advantageous as it avoids the cellular damage as in procedures involving enzymatic digestion. However, none of the methods have been standardized till yet.

Currently, the MSC are being isolated from the Wharton's jelly by collecting the umbilical cord samples followed by rinsing with 70% ethanol and Hanks' balanced salt solution (HBSS). The vessels are removed and the jelly fragmented and plated with complete media. This is followed by 37°C incubation under low oxygen conditions. The tri-gas incubator is used along with the specific composition of humidified gas mixtures. The mediumis replaced after one week and sub-cultured until 80-90% confluency is obtained. The medium is completely removed two times a week and after 14days. 0.25% trypsin- EDTA is used for harvesting the MSC.

IDENTIFICATION OF HSC AND MSC:

Umbilical cord stem cells consist of different types of stem cells population. The researchers have narrowed down into three main types of stem populations from umbilical cord blood, viz.,HSC, MSC and multipotent non-hematopoietic stem cells.

New blood cell formation is known as haematopoiesis.²⁵ Different populations of HSC is found in the umbilical cord. It expresses distinct types of antigens. They are CD133, CD177, CD9, and CD45 at the different stages.²⁰ They are found to be negative for CD34.^{5,26, 27}

In case of MSC, Till now, there is no specific marker for identification of these stem cells. However, the expression of several cell surface antigens like the mesenchymal stem cells derived from umbilical cord are in general identified by the expression of CD10, CD13, CD29, CD44, CD105 etc. and REX2, SOX2, NANOG, ganglioside 2 (GD2) etc.These, along with their ability to differentiate into multiple cell types in vitro,are used for the identification of these stem cells. ^{9, 17, 28, 29} All these markers are not specific and are expressed by other cell types also.³⁰ The CD105 expression is considered to be essential in the mesenchymal stem cells but this is still contradictory.³¹ Mesenchymal stem cells, giving rise to cardiomyocytes, express N-cadherin and cardiac troponin.^17,20 However, this expression is still being debated.

PROLIFERATION OF HSC:

The proliferative properties of HSC are highly regulated by the communicating cytokines. The proliferation includes survival and expansion of the cells and it is regulated by lineage-specific factors that emerge during the later period of proliferation. It includes M-CSF, G-CSF, and IL-5 that evolve during the initial stages of developing cells and controlled by various cytokines.^26,32 When the cells exit from G0 some cytokines like IL-3, GM-CSF and IL-4 control the cell proliferation. Some growth factors are also involved in enhancing the survival and for amplifying the matured cells proliferation.³³Some early acting cytokines like IL-6, G-CSF, IL-11, IL-12, LIF, and SF are involved in the proliferation process.

PROLIFERATION OF MSC:

The proliferation of these stem cells is a long-term ex vivo process.^9,34These stem cells have been found to have a higher proliferative power and doubling rate than the ones isolated from the bone marrow or other sources. ^17,21,23. The UC-MSC also has a greater ability to form colony forming units that is called as fibroblasts. ^35,36

DIFFERENTIATION OF HSC:

The differentiation of HSC is stochastic in behaviorand it comes from the intrinsic properties of the progenitor cells. Some of the receptors found on the mature cells can also direct differentiation of the cells. But this is a controversial discussion as growth factors prevent apoptosis. Ras signaling pathway is involved in the differentiation process.³³ HSC’s differentiate into multipotent stem cell and progenitor cells. Progenitor cells give rise to myeloid precursors. It includes Colony Forming Unit of Granulocyte, Erythroid, macrophage, and megakaryocyte [CFU-GEMM] and CFU-Lymph. CFU-GEMM gives rise to Colony forming unit of erythroid[CFU-E], Colony forming unit of megakaryocytes [CFU-Meg] and colony forming unit of granulocytes and macrophages[CFU-GM].²⁶

DIFFERENTIATION OF MSC:

The umbilical cord-derived MSC can be induced to differentiate into mesodermal and non-mesodermal lineages. These stem cells can give rise in vitro to skeletal muscle cells, smooth muscle cells, osteoblasts, fat cells and chondrocytes etc.^7,28,37,38 These stem cells have also been found to differentiate into the endothelial cells and neural cells upon exposure to proper media.^17,21,34 The potential to differentiate into endothelial cells is higher as compared to the MSC derived from other sources.^1,21 The umbilical cord-derivedMSC that overexpress hepatocyte growth factor have been found to differentiate possibly into the dopaminergic neuron-like cells after being transplanted in rats with collagenase-induced intracerebral haemorrhage.¹⁷ These stem cells can differentiate after myocardial infraction into myocardial like cells and also express the genes such as GATA-4, cTnT, a-actin and Cx43.^17,21 However, the potential of the stem cells also differs based on the particular region of a tissue from which it has been isolated or derived.³⁹

ADVANTAGES:

The advantages of cord stem cells are:

1. They are more proliferative in nature and show less dependency on stromal cells. The high proliferative potential, capable of forming colonies, is present in more number of cord cells.²⁶

2. They do not form teratomas on transplantation and there is no ethical or legal issues in their research.^9,
24

3. They have longer self-renewal properties in vitro and shorter replication period in comparison to the MHC derived from the bone marrow. ^9,24

4. They have high endothelial differentiation potential as compared to MSC derived from the bone marrow sample and also a lower risk of infection.²⁴.

5. Umbilical cord blood is considered as potential source of stem cells for research and clinical applications. It has high plasticity and is abundant in nature because of which it is considered as a rich source of stem cells.⁵

6. For treatment of various malignant diseases like lymphoma and leukemia and nonmalignant diseases like hematological diseases, umbilical cord blood transplantation has been used.⁴⁰

7. Umbilical cord blood has a high population of multipotent stem cells that differentiate into different cell types, including endothelial, myotubes, epithelial and neural. So it can be considered as an alternative of embryonic stem cells.⁴¹

8. Umbilical cord blood cell act as cell replacement source and can also act as neuroprotective, anti-inflammatory agent and .⁴²

9. Umbilical cord blood can be beneficial for the donor as a future source of stem cells.⁴³. It can be helpful in the treatment of blood relations and close relatives.⁴⁴

10. As per the study, it is analyzed that survival rate is higher in patients who undergo umbilical cord transplant than the patients who receive bone marrow transplant.^45,46 In transplantation, multipotent adult stem cell is very beneficial.

DISADVANTAGES:

The disadvantages associated with the use of umbilical cord mesenchymal stem cells are very few. One of the disadvantages is that genomic tests are required to be donein order to confirm the weight of the donor baby which cannot be ascertained otherwise in advance before delivery.²⁴. Less number of cells are obtained per individual cord blood cell limiting the availability of the HLA-match donors.⁵There is also difficulty in storing umbilical cord.⁴³Other disadvantages include lack of clinical experience with umbilical cord blood especially in adults and also the lack of high-quality units and large units in the cord blood bank with an average 350-400000 units stored worldwide.⁴⁷

THERAPEUTIC USES:

Cancer still remains a deadly disease with no ultimate cure all over the world. MSC hold the potential for cell-based therapies in cancer patients.^7,9,48 These stem cells have the potential to destroy the osteosarcoma, breast cancer cells and also the ovarian carcinoma cells in vitro.^17,21,39,49 These stem cells play a role in modulating the microenvironment of the tumor cells and thereby have anti-cancer effects. HSCare also used for the treatment of cancer. In case of the AML patients, allogeneic hematopoietic stem cell transplantation has been done.⁵⁰Many clinical trials have been going on to treat various neurological diseases such as spastic cerebral palsy and hypoxic-ischemic encephalopathy. Currently, for treatment of spinal cord injuries, a clinical trial is on.⁵

Umbilical cord blood provides an arsenal of therapeutic effects in one transplant.⁴²Umbilical cord-derived MSC also hold potential for being used for transplantation in patients with many disorders like Systemic Lupus Erythematosus, hereditary ataxia, spinal cord injury, Parkinson’s disorder, autoimmune disorders like Type1 diabetes.^9,17,21,48It is also being used in the cases involving liver fibrosis. ³⁹ These stem cells can control excess abnormal proliferation by interacting with B-lymphocytes and also the produce antibodies against B-cells itself in larger amounts.^51,52Umbilical cord-derived stem cells may also be used in the treatment of neurological disorders like autism, cerebral palsy, traumatic brain injury etc.⁵³

FUTURE PERSPECTIVES:

Research on HSC will bring in arevolution in the treatment of blood disorders and can be used for transplantation procedures. In future, safe reprogrammed cells can be developed to remove genetically defective HSC and allow the transplantation of the corrected cells against particular diseases.MSC, derived from umbilical cord stem cells are being used for transplantation and holds future potential for preventing the graft versus host disease.^17,55,56 These stem cells will also serve as a tool for future advancement in tissue engineering tools and techniques.^7,28,39 Future application of these stem cells includes widespread use in the treatment of cancer and neurodegenerative disorders.⁴⁹ It may also be used for bio-banking as it does not involve ethical and legal issues in its use and is easy to collect.³⁹ Future work and studies on cord stem cells will help in a better understanding of the stem cells thereby helping in the therapies and treatment of disorders that are still a question mark for the world. More studies and investigations in this direction is a need. Clinical trials are being carried out with umbilical cord blood for treating hematological conditions, diabetes, heart disorders and inborn errors of metabolism.

With the use of umbilical cord blood, many clinical trials are in early stages of developing an effective treatment for injuries and disorders.⁵ HSC and progenitor cells originate from the umbilical cord blood. Umbilical cord blood cells also contain various types of progenitor cells and stem cells.All these hold promise for future treatment and therapies.

CONCLUSIONS:

Stem cells are going to play an important role in the field of medicine. The understanding of stem cells will provide the knowledge with respect to the clinical significance of these cells. Stem cells with the capacity of self-renewal and differentiation hold promise for the treatment and therapies of many disorders that still remain to be a challenge for mankind. The umbilical cord is one of the rich sources of stem cells. Umbilical cord stem cells are easy to obtain and various diseases like cancer, neurodegenerative disease, and many blood-borne diseases can be treated with them. They have many more benefits as compared to other sources of stem cells. In this review, the detailed study of stem cells and their types in a structured manner have been compiled. The isolation, proliferation, identification, and differentiation of HSC and MSC have been elaborated. The advantageous and disadvantageous of these cells have been outlined.However, stem cell research involves many challenges that need to be addressed to get the desired outcome in the future.

ACKNOWLEDGEMENTS:

The authors thank VIT, Vellore for providing the facilities for the preparation of this manuscript.

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Received on 15.02.2018 Modified on 27.03.2018

Research J. Pharm. and Tech 2018; 11(6): 2709-2714.

DOI: 10.5958/0974-360X.2018.00500.0