Human stem cells – sources, sourcing and in vitro methods

Stem-cell therapies are fast becoming a key instrument in treating many diseases, due to a large amount of trials to implement them in clinical setting. A milestone in this field research was achieved when mice ICMs were isolated by Martin et al. in 1918 and formed a teratocarcinoma when injected subcutaneously into host [29]. Many authors were inspired by this accomplishment, which contributed to new attempts in this area. This resulted in a proof that cells present in teratocarcinomas are pluripotent and capable of differentiating into at least two lineages [51]. Another vital step was made when Thomson and associates, who isolated 14 inner cell masses from five separate human embryos in blastocyst stage. These five ES cell lines maintained a potential to differentiate into all three embryonic germ layers [2]. Recent studies suggest that naive hESCs can be converted into cells with a transcriptome and methylome similar to human trophoblast stem cells (hTSCs). This allowed core placental gene reactivation and placenta-like methylome acquisition [52].

HSCs constitute 1% of bone marrow mononuclear cells (BMMNCs) [53], with their transplantation being a common procedure intended to treat many diseases such as leukemia, lymphoma or myeloma. There have also been numerous studies to investigate whether there are different types of stem cells in bone marrow, which could be used in the stemcell therapy. For example, endothelial progenitor cells (EPCs) were isolated from bone marrow to treat ischemic stroke through EPCs transplantation [54]. Moreover, there are stem cells in bone marrow and peripheral blood which may play more significant role in vasculogenesis than EPCs. It was proved that the VSELs exhibit endothelial phenotype in vitro in the presence of VEGF-B, as well as in vivo in Matrigel implants [55].

It is now well established that adipose-derived mesenchymal stem cells (AMSCs) can be used in plenty of therapeutic procedures. Abdominal fat tissue, which can be collected by lipoaspiration, may be considered as a crucial source of AMSCs. Therefore, designing the scaffold-free three-dimensional (3D) grafts made of these cells is increasingly plausible [56].

A large and growing body of literature has also investigated the relevance of epidermal stem cells, which can be divided into dermal-derived neural crest stem cells, MSC-like dermal stem cells and dermal hematopoietic cells [57]. Furthermore, in other studies self-renewal and multipotency of two different types of stem cells in each hair follicle were revealed. These cells formed epidermis and hair when grafted into mice [58].

Several attempts have been made to prove that limbal epithelium can be considered as an essential source of stem cells. The surgical techniques of limbal transplantation improved over time, with this procedure requiring less donor tissue not needing a specialist laboratory to harvest cells [59].

In recent years, there has been an increasing amount of literature focusing on stem cells, which can be found in human oral cavity. For example, dental pulp stem cell (DPSC) isolation demonstrated that these cells have the ability to form dentin and pulp-like complex [60]. This discovery contributed to many transplantation trials, which revealed that mobilized dental pulp stem cells (MDPSCs) have therapeutic potential to regenerate the pulp in pulpitis [61]. Other authors investigated the presence of human stem cells in periodontal ligament and showed their ability to differentiate into cementoblast-like cells, adipocytes, and collagen-forming cells. When injected into immunocompromised rodents, these cells contributed to periodontal tissue repair [62]. Human mucosa also seems to be a promising source of stem cells for autologous transplantation therapies. Although these structures were isolated from oral mucosal epithelium or even from adult olfactory mucosa, many clinical trials are needed before they will be used in common treatment procedures [63]. Moreover, recent studies also highlight that stem cells can be easily collected from voided urine. Human-urine-derived stem cells (USCs) are capable of differentiating into osteogenic, chondrogenic, adipogenic, and neurogenic lineages [64].

An easily accessible source of stem cells, which can be used for autologous transplantations, has been a subject of scientists’ interest for years. Studies analyzing if there are MSCs in human placenta seem promising, with one of the first trials revealing the presence of these cells and their ease of isolation from the mentioned structure. Moreover, PMSCs were cultured using a very simple technique, without characteristic morphological changes in medium [65]. Another source of stem cells, which can be obtained non-invasively, may be the human umbilical cord. These cells seem to have a great potential not only to differentiate into many structures but also regulate monocyte secretome [66]. Isolating cells from Wharton’s jelly may also be a simple method of obtaining MSCs. It has been suggested that they can be easily harvested and have properties to initiate the process of wound healing [67]. Recently, there have been several reports showing the presence of stem cells in amniotic fluid, with isolated cells capable of differentiating into multiple lineages such as hepatic, neuronal, myogenic, adipogenic, osteogenic and endothelial. Additionally, neuronal lineage presented an ability to secrete neurotransmitter L-glutamate and express G-protein-gated rectifying potassium channels [68].

Mouse adipose-derived mesenchymal stem cells (mADMSCs) have been used in the therapy of liver fibrosis (LF) induced by carbon tetrachloride (CCl4) in a mouse model. mADMSCs were injected into the tail vein once a week for 2 weeks. Moreover, mADMSCs with antioxidant preconditioning could improve restoration of liver function and expand the intrahepatic transplantation of mADMSCs [69].Furthemore, mADMSCs have been used in treatment of the mouse model of chronic ovalbumin-sensitized asthma, through endotracheal administration at 21^st day of disease. Therapy with mADMSCs could moderate inflammation of the respiratory tract, palliate airway excessive responsive and expand airway remodelling [70]. Furthemore, mADMSCs have been used as a therapy in cutaneous leishmaniasis in a mouse model, through hypodermic injection at the infection surface. The administration of mADMSCs was repeated 4 times more, once a week until 28^th day after infection. The mADMSCs could not remove Leishmania major but briefly facilitated intensification of immune reaction against L. major by upregulating the inflammatory reaction and slowing propagation of infectants [71].

Rat adipose-derived mesenchymal stem cells (rADMSCs) have been used as a therapy in monocrotaline-induced pulmonary arterial hypertension in a rat model. rADMSCs were injected into the jugular vein on the 14^th day of disease. After 2 weeks, lung vascular rebuilding was mitigated, which expanded hemodynamics [72]. Moreover, undifferentiated rADMSCs (u-rADMSCs) have been used in therapy following partial hepatectomy in a rat model. u-rADMSCs were administered via the portal vein or into the parenchyma immediately after liver resection. They could stimulate the liver recovery and moderate the histopathological liver insult by concurrently expanding the expression of specific regeneration-genes for liver, regardless of their number and the way of transplantation [73].

Bama pig adipose-derived mesenchymal stem cells (BpADMSCs) have been used in the therapy of LF caused by CCl4 in a mouse model. After 4 weeks of treating the mice with CCl4, BpADMSCs were administered through the portal vein. This improved liver regeneration, as well as reduced necrosis, inflammation and fibrosis [74].

Human adipose-derived mesenchymal stem cells (hADMSCs) associated with a scaffold of collagen sponge have been used to construct a full-thickness skin graft in a mouse model. The application of full thickness graft of tissue-engineered skin improved wound treatment and encouraged injure repair at soon as 7 days after the surgery [75].

Mouse bone marrow mesenchymal stem cells (mBM-MSCs) have been used in the therapy of mouse oxygen-induced retinopathy model by injection into the vitreous body. Observation for 10 days after transplantation showed that the mBM-MSCs could suppress neovascularization and present a preventive effect on retinopathy induced by oxygen. Moreover, the mBM-MSCs could migrate to the site of damage on retina, reduce the apoptosis of retinal cells and expand the density of retinal vessels [76]. Furthermore, microRNA-9-5p modified mBM-MSCs (miR-9-5p-m-mBM-MSCs) have been used in bone cancer pain therapy in a mouse model. The miR-95p-m-mBM-MSCs were infused through a catheter placed in the subscleral region each day from 2 days before implantation of murine sarcoma cells until 21^st day after surgery. miR-9-5p-m-mBM-MSCs could mitigate pain caused by bone cancer by regulating inflammation of the nervous tissue in the central nervous system [77]. The mBM-MSCs have been induced for endothelial differentiation and used as a therapy in hindlimb ischemia in a mouse model. The endothelial-like cells (ECs) were injected into the paracentral thigh of the gastrocnemius muscle in 3 various places within 1 day after the model surgery. The ECs could increase therapeutic result of angiogenesis and have stronger ability in vascular creation [78].

Rat bone marrow mesenchymal stem cells (rBM-MSCs) have been used in the therapy of LF induced by CCl4 in a rat model. The rBM-MSCs were injected via the penile vein after one month treatment with CCl4 in two doses, the second one week after the first. The rBM-MSCs were able to reconstruct liver architecture and soften CCl4 induced LF [79]. The rBM-MSCs have been used in healing of peripheral nerve damage and restoring limb function in a rat model. The rBM-MSCs injections were administered at the nerve surfaces needing repair during the surgery. After the procedure, the rBM-MSCs were injected in the dorsal penile vein and readministered for 3 weeks with one-week interspace between doses. The rBM-MSCs could improve restoration of extremity function and promote the nerve recovery [80]. The rBM-MSCs have been used in diabetic ischemic wound treatment on the dorsal region of foot of noninsulin-dependent diabetes mellitus in a rat model. The rBM-MSCs were injected locally, intracutaneously or by infusion through the left femoral vein, instantly after the wound was created. After 14 days, both local and systemic administration of rBM-MSCs stimulated the wound treatment process. Moreover, the systemic infusion could remedy the injured tissue and mitigate hyperglycemia [81].

The transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) has been used in a treatment of allergic rhinitis in a mouse model. The hUC-MSCs were injected several times at specifies intervals, intraperitoneally or into the tail vein. The hUC-MSCs migrated to the nasal cavity and suppressed the allergic reaction. However, tail vein injection was more effective in this therapy [82]. Furthermore, hUC-MSCs have been used in the therapy of collagen-induces arthritis in a rat model. The injection of hUC-MSCs was administered through the tail vein on 10^th day, decreasing the injury of bone in the ankle joints after 28 days and exhibiting therapeutic effect through modulation of intercommunication among gut flora and host resistance via the aryl hydrocarbon receptor [83]. Moreover, the freshly defrosted hUC-MSCs (D-hUC-MSCs) and cultured hUC-MSCs (C-hUC-MSCs) were administered in a case of full-thickness flaw of rotator cuff tendon in a rat model. The D-hUC-MSCs or C-hUCMSCs were injected in two separated doses into the tendon approximal at both sides of the injury, which resulted in regeneration of tendons comparable to C-hUC-MSCs [84].

Human chorionic plate-derived mesenchymal stem cells (hCPDMSCs) have been used in therapy of cyclophosphamide-induced premature ovarian insufficiency in a mouse model. The hCPDMSCs were administered by the tail vein one time per week for 4 weeks. The transplantation of hCPDMSCs improved the function of ovary and enable follicle and oocyte growth [85].

Gingival mesenchymal stem cells (GMSCs) have been obtained from human fetal gingival tissue and used in gingival defect therapy in a rat model. The GMSCs were grafted into the injury area of the gingiva 6 days after excision of the tissue around the mandibular incisor. The GMSCs were able to reduce the imperfection area 1 week after grafting, also recreating the color and morphology of local tissue, resembling natural gingiva, 3 weeks after transplantation [86].

Human mesenchymal stem cells isolated from Wharton’s jelly (hW-MSCs) have been induced to endothelial differentiation and used as a therapy in aged diabetic rat model. The trans-differentiated ECs (tdECs) were injected by the tail vein 6 weeks after diabetes verification. The tdECs derived from hW-MSCs could counter the dysfunction of endothelium, inflammation and oxygen-induced stress [87].

Stem cells from human exfoliated deciduous teeth (SHED) combined with a scaffold of hydroxyapatite have been grafted to into a defect of alveolar bone in a rat model. The SHED with hydroxyapatite scaffold could be an efficient agent in the regeneration of defects of alveolar bone by raising osteoprotegerin and decreasing expression of receptor activator of nuclear factor-kappa B ligand [88].

The research on embryonic stem cells started in the 1950s. In 1954, spontaneous testicular teratoma was discovered in mouse strain 129. This discovery started experimental work on teratomas and teratocarcinomas. Leroy C. Stevens and C.C. Little proved that teratomas are composed of many types of embryonic and adult tissues that are not typically found in testis. They transplanted fifteen spontaneous teratomas and one of these developed into a transplantable tumour which consisted of undifferentiated cells with the ability to grow subcutaneously and in ascitic fluid [89].

Another important study showed the multipotentiality of single embryonal carcinoma cells. The study involved the transplant of over 1700 single cell grafts. As a result of transplantation, 44 clonal lines were obtained, 43 of which were teratocarcinomas composed of different well-differentiated somatic tissues [90].

Interest in this subject increased in 1970s, due to improvements of knowledge on mammalian development and cell differentiation. Martin and Evans subcloned mass cultures of pluripotent teratocarcinoma cell lines, which led to the discovery of the cells with large nuclei and dark cytoplasm that grew in the form of small colonies. These cells could proliferate and produce teratocarcinomas after subcutaneous injection, and became known as embryonal carcinoma cells [91, 92].

Mouse and human embryonal carcinoma cell lines were identified by specific markers, characteristic for the embryonal carcinoma cell and disappearing during differentiation. The first marker was the alkaline phosphatase enzyme. Later, thanks to the development of the monoclonal antibodies, several specific proteins were discovered. The antibodies detecting mouse embryonal carcinoma cells react with the Forssman antigen and stage-specific embryonic antigen 1 (SSEA1). Furthermore, it turned out that mouse embryonal carcinoma cells express SSEA3 and SSEA4 during differentiation. On the other hand, human embryonal carcinoma cells express SSEA3 and SSEA4, and SSEA1 only during differentiation [92].

Similarities between mouse embryonal carcinoma cells and the cells of early embryos were also noticed. This and the fact that teratocarcinomas can be derived from embryos led to further research on whether embryonal carcinoma cells retain their embryonic nature. Teratocarcinoma cells were transferred into a blastocyst and, after the blastocyst development, small colonies of the transferred cells were visible in the embryo, some of which persisted into the adult [93].

The knowledge gained by studying teratocarcinomas and embryonal carcinoma cells resulted in the development of embryonic stem cells (ES). In 1981 Evans, Kaufman and Martin discovered that the cells from early mouse embryos that are exposed to the same environment can stop development and continue to multiply [94].

Human embryonic stem cells (hES) were isolated from the human blastocyst for the first time in 1998. These cells could form trophoblast and derivatives of all three embryonic germ layers after 4-5 months of proliferation in vitro [2]. In the same year, hES were obtained from human embryonic germ cells (hEG), isolated from gonadal ridges and mesenteries of 5–9-week-old human fetuses from abortions performed for health reasons [95]. These procedures of obtaining hES lead to the destruction of the embryo, which causes serious ethical problems. Therefore, attempts were made to invent other techniques for derivation of ES cells. In 2006, hES cells were derived from single blastomeres [96], with this procedure not disturbing the further development of the embryo. Other sources of hES cells turned out to be arrested embryos [97] and parthenogenetic blastocysts [98].

The next achievement was the induction of pluripotent stem cells from differentiated somatic cells. These cells were named induced Pluripotent Stem cells. The first iPS cells were obtained from mouse embryonic and adult fibroblast cultures by introducing specific factors - Oct3/4, Sox2, c-Myc and Klf4 [3]. A year later, the same experiment was performed using human somatic cells, which proved that iPS cells can be generated from adult human fibroblasts [3].

There are three main factors responsible for maintenance of pluripotency of ES cells: Oct3/4, Nanog and Sox2. These factors create the regulatory network to control each other’s expression levels [99]. Oct4, Nanog and FoxD3 form a negative feedback loop in order to maintain their expression in pluripotent ES cells [100]. Oct3/4 and Sox2 genes are involved in a synthesis of transcription factors such as UTF-1, REX-1/Zfp-42, telomerase reverse transcriptase (TERT) and telomeric repeat binding factor 1 and 2 (TERF1, TERF2).

Reprogramming of cells has great potential for medical applications. However, iPS cell form teratoma when injected into tissues. Hence, the process of transdifferentiation seems to be an interesting solution. Transdifferentiation is a transformation of one specialized cell into another. Pluripotent cells don’t take part in this process, so it doesn’t carry the risk of teratomagenesis [101]. An example of transdifferentiation is the study from 2008, which involved reprogramming of adult mice pancreatic exocrine cells to beta-cells using three transcription factors - Neurog3, Pdx1 and Mafa [102]. Another finding showed that human fibroblast can be reprogrammed to cardiac-like myocytes. This procedure included inducing expression of cardiac markers in neonatal and adult human fibroblasts [103].

All of these studies indicate that bioengineering methods using cells can have great application in clinical medicine.

Stem cells are increasingly used nowadays in clinical practice due to their unique properties. They are employed in studies in the field of regenerative medicine, orthopedics, cardiology, neurology and many others.

The employment of stem cells in regenerative medicine enables very effective treatment and satisfactory results. Gjerde et al., in their trial, used bone marrow- derived MSCs to induce regeneration of mandibular bone defect, which is a less invasive method than autologous bone grafting, a current gold standard procedure. BCP (biphasic calcium phosphate) granules, used as scaffold material, with MSC’s attached, were introduced into the operation site. There was noticeable increase in both volume and width of the alveolar ridge. Moreover, all patients were pleased with the clinical outcome of the procedure, which was yet another crucial benefit of this method [104]. Stem cells have also proved useful in skin wound healing process, providing better aesthetic outcomes, but most importantly solving the problem of ineffective vascularization. For this purpose, Samberg et al. applied combination of PEGylated PRP (platelet rich plasma) hydrogel and ASCs (adipose-derived stem cells). High concentration of growth factors contained in platelets and fibrin-made scaffold created optimal conditions for effective angiogenesis and new tissue’s growth. Using hydrogel turned out to be an effective way to maintain moist environment within the wound. This approach was tested in vitro and on an animal model. The study showed that applying this method resulted in greater blood vessel density in PRP + ASCs group than in the others (without PRP or ASCs). While amendment of other parameters, such as faster wound closure, was not observed, it is a potentially efficient procedure that could be considered when it comes to an improvement of wound healing [105]. In other study allogenic ASCs were applied in a form of hydrogel-based sheets to patients with diabetic foot ulcers. In the treatment group, wounds healed faster and more patients achieved complete wound closure compared to the control group [106]. Stem cells could also be used for regeneration and preservation in reproductive medicine. Jia et al. used human umbilical cord stem cell conditioned medium (hUC-MSC-CM) to reduce cryoinjury in frozenthawed human OCTs (ovarian cortical tissues). In the hUC-MSC-CM group, neoangiogenesis was increased and follicular apoptosis was decreased in comparison with the SF-CM (serum- free culture medium) group. This effect is in part assigned to hUC-MSC’s paracrine activity, with growth factors and cytokines acting as protective factors. This method may be used in the future as an effective way to reduce ischemic damage and apoptosis of follicles in OCTs and, as a result, to avert fertility loss [107].

Stem cells are becoming increasingly interesting, not just when it comes to regenerative medicine. Nowadays, SCs are also often employed in orthopedics. Gupta et al. used bone marrow-derived, cultured, pooled, allogenic mesenchymal stromal cells (Stempeucel ®) to treat osteoarthritis, which is a type of joint disease involving degeneration of joint cartilage. Intra- articular injection of Stempeucel ® + HA resulted in significant pain reduction on an animal model in comparison with a group that was treated only with HA, with improvement in cartilage repair also observed [108]. In other study, Lamo – Espinosa et al. applied intra-articular injection of autologous bone marrow MSCs to patients with osteoarthritis. Those who were given BM-MSC noticed major pain reduction, while others who were given HA did not report any significant changes. There was also an improvement in the range of knee motion in the group who had BM-MSC injection (especially- higher dose). These results suggest that therapies using MSCs are safe and may be used more often in the future of orthopedics [109].

Nowadays, an increasing number of methods using stem cells are being tested as a treatment for patients with heart failure (HF) with low ejection fraction. Intravenous infusion of umbilical cord mesenchymal stem cells (UC-MSC) was used by Bartolucci et al. as a therapy for patients with HF. Improvement in left ventricular ejection fraction (LVEF) and quality of life was observed in UC-MSC group [110]. In other study, Soetisna et al. used autologous CD133+ bone marrow stem cells to examine their impact on cardiac function. Transepicardial and transseptal implantation of said cells was performed on patients with coronary heart disease with low ejection fraction during coronary artery bypass grafting. There was a significant increase in LVEF and decrease in WMSI (wall motion score index) in CD133+ group compared with control groups. Improvement was also noticed in scar size proportion in CD133+ group. These results suggest that this treatment may be applied for better cardiac repair and patient’s better life quality [111].

Stem cells have been also used by Oh et al., who applied autologous bone marrow-derived mesenchymal stromal cells to patients with amyotrophic lateral sclerosis (ALS). MSCs were administered intrathecally in two succeeding injections. The decline in ASLFRS-R score was not accelerated, furthermore it was more gradual during the follow-up in comparison with the lead-in period. Moreover, after MSC injection IL-10 and TGF-β levels were elevated, while MCP-1 level was decreased, which suggests that this treatment may be beneficial for ALS patients’ immune response [112]. Similar results were achieved in other clinical study with MSC application in ALS patients [113], which indicates that MSC administration could be an effective ALS treatment in the future.

Pang et al. used allogenic bone marrow-derived mesenchymal stromal cells expanded in vitro as a possible approach of treatment for patients with aplastic anemia (AA). MSC’s time to response (TTR) was significantly shorter than TTR of immunosuppressive factors that are used in AA therapy. The improvement of leukocytic and megakaryocytic lineage was also observed - after the treatment white blood cell counts, platelet counts, hemoglobin counts and Treg percentages were considerably higher in comparison to the baseline. This suggests that while the treatment is potentially a good solution for patients suffering from AA, it requires further evaluation [114]. Peripheral blood stem cell (PBSC) transplantation can be used in the treatment of AA as well. In their trial, Purev et al. used PBSC allograft containing CD34+ cells and non-mobilized T-cells in lower dose than in unmanipulated PBSC. This approach also resulted in neutrophil and platelet recovery. What is more, the risk of acute and chronic GVHD was decreased compared to unmanipulated PBSC [115].

In a recent study, Sengupta et al. attempted to treat patients with severe COVID-19 with exosomes (ExoFlo) derived from BM-MSC. Patients’ oxygenation improved after administration suggesting that ExoFlo may prevent oxygenation’s deterioration, and consequently the necessity of mechanical ventilation. Inflammatory markers (CRP, ferritin, D-dimer) were decreased and there was a major improvement in neutrophilia and lymphopenia observed as well [5]. In turn, Leng et al. transplanted MSCs to patients with COVID-19 pneumonia. Patients’ clinical status improved significantly after MSC administration- CRP, TNF-α decreased, whereas IL-10 and VEGF rose, which promoted patients’ recovery. MSCs are known to have immunomodulatory effect; therefore, they could prevent cytokine storm which is a highly dangerous incident during COVID-19. This therapy turned out to be particularly effective for critically severe patients [116].

Dhere et al. used autologous noncryopreserved BM-MSC as a novel therapy for patients with moderate to severe Crohn’s disease. This method turned out to be safe and well tolerated by participants. 5/11 of enrolled patients responded to the treatment and 6/11 did not. What is more, allogenic MSCs would pose a risk of immune rejection, thus application of autologous MSCs was a safer solution. The results are promising, but estimation of this method’s efficacy requires further research [117]. In other clinical trial Zhang et al. reported that UC-MSC infusion is a potentially effective treatment for patients with Crohn’s disease. The results showed noticeable improvement in patients’ clinical status. Crohn’s disease activity index declined as well as Harvey-Bradshaw index and corticosteroid dosage a year after the treatment. It was also noticed that UC-MSC administration was safe in general, with only mild side effects such as fever occurring. However, long-term effect and mechanism of action of UC-MSC treatment is not known and is yet to be studied [118].

MSCs were used by Zhao et al. to treat patients with acute graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation. Majority of patients responded to the treatment which is a promising result that is ascribable to MSC’s immunomodulatory activity. Patients from MSC group presented an increased Treg count and decreased incidence of chronic GVHD compared to the control group. There was also thymic function improvement noted, evaluated by observing changes in sjTREC (signal joint T cell-receptor excision DNA circle) levels. Due to these mechanisms MSCs might reduce severity of chronic GVHD without increased risk of infections or tumor relapse [119]. In other trial, single-dose MSCs were applied to patients before haploidentical peripheral blood stem cell transplantation (PBST). This treatment resulted in a decline of incidence of severe acute GVHD in MSC group compared to the control group [120].

Carlsson et al., in their study, researched safety and efficacy of autologous MSCs applied to patients with new-onset type 1 diabetes. In MSC group, C-peptide response to mixed meal tolerance test did not decrease. Moreover, in some cases it was elevated with no side effects reported. The results suggest that this treatment may be an effective way to preserve β-cell function, although it requires further examination [121]. In other study, issue of autoimmune activity of T-cells in type 1 diabetes was addressed through the use of use of Stem Cell Educator (SCE) which is a therapy that employs human multipotent cord-blood stem cells (CB-SC). Lymphocytes separated from patient’s blood are transported to the SCE device and after interaction with CB-SC educated lymphocytes are returned to patient’s bloodstream. The outcome indicates that this therapy modifies patient’s immunological response and helps to restore immune system balance [122].