Umbilical Cord Stromal Cells Research Articles

Bovine Fibroblast-Derived Extracellular Matrix Promotes the Growth and Preserves the Stemness of Bovine Stromal Cells during In Vitro Expansion

Cultivated meat is a fast-growing research field and an industry with great potential to overcome the limitations of traditional meat production. Cultivated meat utilizes cell culture and tissue engineering technologies to culture a vast number of cells in vitro and grow/assemble them into structures mimicking the muscle tissues of livestock animals. Stem cells with self-renewal and lineage-specific differentiation abilities have been considered one of the key cell sources for cultivated meats. However, the extensive in vitro culturing/expansion of stem cells results in a reduction in their abilities to proliferate and differentiate. Extracellular matrix (ECM) has been used as a culturing substrate to support cell expansion for cell-based therapies in regenerative medicine due to its resemblance to the native microenvironment of cells. In this study, the effect of the ECM on the expansion of bovine umbilical cord stromal cells (BUSC) in vitro was evaluated and characterized. BUSCs with multi-lineage differentiation potentials were isolated from bovine placental tissue. Decellularized ECM prepared from a confluent monolayer of bovine fibroblasts (BF) is free of cellular components but contains major ECM proteins such as fibronectin and type I collagen and ECM-associated growth factors. Expansion of BUSC on ECM for three passages (around three weeks) resulted in about 500-fold amplification, while cells were amplified less than 10-fold when cultured on standard tissue culture plates (TCP). Moreover, the presence of ECM reduced the requirement for serum in the culture medium. Importantly, the cells amplified on ECM retained their differentiation abilities better than cells cultured on TCP. The results of our study support the notion that monolayer cell-derived ECM may be a strategy to expand bovine cells in vitro effectively and efficiently.

Transdifferentiation of Human Umbilical Cord Stromal Cells to Neurogenic-Like Cells

Objective: To investigate the differentiation of human umbilical cord stromal cells into neurogenic cells. Methods: (1) Human umbilical cord stromal cells (HUMSCs) were pre-induced with recombinant human fibroblast growth factor-2 (hbFGF-2, Peprotech, UK) and N² (1:100, Invitrogen, USA), immunocytochemistry and Western blot to determine the expression of nestin and fibronectin before and after pre-induction. Western blot to identify Nestin and fibronectin expression before and after pre-induction. (2) The differentiation of pre-induced and un-induced HUMSCs into neurogenic cells was performed, and the value-added rates of both were measured by MTT at different time points, and the morphological differences between the differentiated cells and the differentiated cells were verified at the gene and protein levels. Results:(1) 94.4 ± 2.3% and 6.5 ± 1.2% HUMSCs expressed ectodermal marker-fibronectin and stem cell marker-nestin, respectively, and 62.3 ± 5.2% piHUMSCs showed nestin immunopositive staining after adding FGF2 and N² for pre-induction; 51.3 ± 5.9% of cells showed fibronectin immunopositive staining. piHUMSCs showed significantly higher nestin positivity than HUMSCs (P<0.05), whereas HUMSCs expressing fibronectin showed significantly higher positivity than piHUMSCs (P<0.05). Western blot results were consistent with cellular immunohistochemistry results. The proliferation rate of HUMSCs and piHUMSCs by MTT assay suggested that after 48 h of culture, both HUMSCs and piHUMSCs entered the logarithmic cell growth phase, and the cell expansion rate of HUMSCs was significantly higher than that of piHUMSCs after 72 h and 96 h of culture.(2) The results of RT-PCR experiments indicated that differentiated human umbilical cord neural stem cells expressed The expression of the corresponding neural stem cell marker genes was statistically different (p<0.05) than that of undifferentiated umbilical cord stromal cells and terminally differentiated neural cells after induction of fractionation. Conclusion: Umbilical cord-derived stromal cells pre-induced with recombinant human fibroblast growth factor-2 (hbFGF-2, Peprotech, UK) and N² (1:100, Invitrogen, USA) were better able to differentiate into neural stem cells with stable expression of cell morphological changes, proteins and genes.

Directed Differentiation of Neural Cells from Human Umbilical Cord Stroma-derived Neural Stem Cells

Objective to investigate the directed differentiation of human umbilical cord stromal-derived stem cells into neural cells. Methods: (1) Human umbilical cord stromal cells (HUMSC-NSCs) were induced to differentiate from primary human umbilical cord stromal cells, and two different methods were used to induce differentiation to neural cells, which were observed under electron microscope. (2) The neural cells induced to differentiate by different induction methods were identified by morphological differences, immunohistochemistry, and Western blot. Results: (1) Some of the suspended NSCs started to adhere to the wall 48-72 h after the addition of the inducer, and elongated protrusions could be seen in 5-7 days with the neurosphere as the center, and cells gradually migrated outward from the neurosphere. 10 days later, several cells with different morphologies could be seen. Some of the cells had increased refractive index and the length of the protrusions increased, showing bipolar growth. In some cases, the protrusions were short and dense, centered on the cytosol, and protruding in a discrete manner. Under electron microscopy, the neurospheres consisted of multiple clonal clusters and two different morphologies of cells, shaped like neurons and astrocytes, could be clearly observed. The number of bipolar neurons was significantly increased in the group with the addition of BDNF.(2) Before induction, 84.5 ± 1.6% and 88.62 ± 1.1% of HUMSC-NSCs expressed stem cell-specific markers: Stro-1 and nestin; neurospheres still expressed 62.7 ± 3.9% of HUMSC-NSCs positive for nestin after 5 d of culture in neuronal cell induction medium. After 10 days of induction, the percentage of immunohistochemically positive stained cells was counted. With the BDMF induction protocol, 38.6 ± 2.9% and 8 ± 1.9% Hoechst33342 positive cells expressed immature (β-tubulin III) and mature (MAP2ab) neuronal cell markers, respectively. Also, 15.8 ± 4.5% and 20.6 ± 4.6% of Hoechst33342-positive cells expressed GFAP (astrocyte marker) and GalC (oligodendrocyte marker). Comparison using a paired t-test revealed that the percentage of β-tubulin III (P<0.001) and MAP2ab (P<0.05) positive cells was significantly higher in the BDNF-induced group than in the general induction group. Western blot results confirmed the immunohistochemical data. Conclusion: After adding BDNF to induce differentiation in the culture group, the number of bipolar neurons was significantly increased, which could promote the induced differentiation of human umbilical cord-derived neural stem cells into neuronal cells.

Wharton\u2019s jelly mesenchymal stem cell-based or umbilical vein endothelial cell-based serum-free coculture with cytokines supports the ex vivo expansion/maintenance of cord blood hematopoietic stem/progenitor cells

BackgroundThe umbilical cord blood (UCB) has been widely accepted as an alternative source of hematopoietic stem/progenitor cells (HSPCs) for transplantation, and its use in adults is still restricted because of low absolute numbers. To overcome this obstacle, expansion of UCB-HSPCs under feeder cell-based coculture is a promising possibility. In this study, we explored UCB-CD34+ cells ex vivo expansion using Wharton’s jelly mesenchymal stem cells (WJ-MSCs) or umbilical vein endothelial cells (UVECs) as feeder layer-based serum-free coculture system with a cocktail of cytokines.MethodsUCB-CD34+ cells were cultured in five different coculture conditions composed of umbilical cord stromal cells (WJ-MSCs or UVECs) with or without a cocktail of cytokines (SCF, FLT3L, and TPO). The cultured cells were harvested at day 10 and analyzed for phenotypes and functionalities, including total nuclear cells (TNCs), CD34+ cells, CD34+CD38− cells, colony-forming unit (CFU) for committed progenitors, and long-term culture initiating cells (LTC-ICs) for HSPCs.ResultsOur work showed the numbers of TNC cells, CD34+ cells, and CD34+CD38− cells were expanded under five coculture conditions, and the feeder layer-based cocultures further promoted the expansion. The numbers of colonies of CFU-GM, CFU-E/BFU-E, and CFU-GEMM in the cocultures with cytokines were significantly higher than their counterparts at day 0 (p < 0.05), while no significant difference (p > 0.05) in those without the addition of cytokines. The numbers of LTC-ICs were increased both under the WJ-MSCs and UVECs with cytokine cocultures, but only in the UVECs group showed a significant difference (p < 0.05), and were decreased under conditions without cytokine (p < 0.05).ConclusionOur data demonstrate that both WJ-MSCs and UVECs as feeder layer could efficiently support the expansion of UCB-CD34+ cells in synergy with SCF, FLT3L, and TPO under serum-free culture condition. The UVECs combined with the 3GF cytokine cocktail could maintain the growth of LTC-ICs derived from UCB-CD34+ cells and even expand to some extent.

Histological Study of Bone Marrow and Umbilical Cord Stromal Cell Transplantation in Regenerating Rat Peripheral Nerve.

Bone marrow and umbilical cord stromal cells are multipotential stem cells that have the ability to produce growth factors that play an important role in survival and generation of axons. The goal of this study was to evaluate the effects of the two different mesenchymal stem cells on peripheral nerve regeneration. In this experimental study, a 10 mm segment of the left sciatic nerve of male Wistar rats (250-300 g) was removed with a silicone tube interposed into this nerve gap. Bone marrow stromal cells (BMSCs) and human umbilical cord stromal cells (HUCSCs) were respectively obtained from rat and human. The cells were sepa- rately cultured and transplanted into the nerve gap. The sciatic nerve regeneration was evaluated by immunohistochemistry, and light and electron microscopy. Moreover, histo- morphology of the gastrocnemius muscle was observed. The nerve regeneration in the BMSCs and HUCSCs groups that had received the stem cells was significantly more favorable than the control group. In addition, the BM- SCs group was significantly more favorable than the HUCSCs group (P<0.05). The results of this study suggest that both homograft BMSCs and het- erograft HUCSCs may have the potential to regenerate peripheral nerve injury and transplantation of BMSCs may be more effective than HUCSCs in rat.

Secreted trophic factors of Human umbilical cord stromal cells induce differentiation and neurite extension through PI3K and independent of cAMP pathway

Trophic factors (TFs) play important role during development and adult tissue maintenance. In neurodegenerative diseases (ND) TF supplementation provides protection. Stromal cells (HUMS) derived from the human umbilical cord matrix provide neuroprotection in the ND models of mice. Though TF mediated protection is known, the exact mechanism of protection is not clear. So, here the essential TFs (secreted by HUMS cells) and the pathway of induction of neurite extension, differentiation and networking is addressed. The HUMS cells from the human umbilical cord matrix were derived and the mouse spinal cord motor neuron cell line, NSC-34 was extensively used. Flow cytometry, immunohistochemistry, RT- PCR, western blot, ELISA and antibody/inhibitor treatment were carried out to figure out the TF pathway. The HUMS cells secrete six neurotrophic factors (sTFs), namely, NT-3, NGF, BDNF, VEGF, IGF-1 and GDNF (TFs). These TFs are sufficient to induce differentiation, neurite extension and neural networking in a motor neuron cell line, NSC34. All the 5 TFs need to be neutralized simultaneously with their antibodies to abrogate neurite extension. These motor neurons express the concomitant receptors, which are either receptor tyrosine kinase (TrK) coupled or to the receptor followed by the TrKs, for the above trophic factors (except for BDNF). The tyrosine kinase inhibitor, K252a, drastically reduces neurite extension. In NSC34, the TFs are coupled to the PI3K-Akt-pathway and the RAS-MAP kinase signaling through phosphorylation of ERK1 and ERK2. PI3K inhibitor, Ly 294002, abolishes neural differentiation and neurite extension. Thus, differentiation, neurite extension and networking could be achieved through the PI3K pathway. Intriguingly, the cAMP second messenger system coupling was not required. H89, PKA-inhibitor caused extensive cell death. But, had no effect in the presence of HUMS-secreted-TFs(HSTFs) suggesting a pathway switch for cell survival itself. HUMS cells and their secreted factors could be of great use in regenerative medicine (RM). The activators of PI3K pathway, the major route of these HUMS-TFs action could be explored in RM and in the neurobiology of neural differentiation and extension.

Clinical neurorestorative progress in traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of death and disability from trauma to the central nervous system. Besides the surgical interventions and symptomatic management, the conventional therapies for TBI and its sequelae are still limited. Recently emerging evidence suggests that some neurorestorative treatments appear to have a potential therapeutic role for TBI and improving the patient's quality of life. The current clinical neurorestorative strategies available in TBI include pharmacological treatments (recombinant human interleukin-1 receptor antagonist, amantadine, lithium, and valproate), the neuromodulation treatments (repetitive transcranial magnetic stimulation, transcranial direct current stimulation, and low-level laser therapy), cell transplantation (bone marrow stromal cells and umbilical cord stromal cells), and combined neurorehabilitation. In this review, we summarize the recent clinical neurorestorative progress in the management of neurodegeneration as well as cognitive and motor deficits after TBI; indeed further clinical trials are required to provide more robust evidence.

Anticancer properties of peptide fragments of hair proteins.

The primary function of hair and fur covering mammalian skin is to provide mechanical and thermal protection for the body. The proteins that constitute hair are extremely resistant to degradation by environmental factors. However, even durable materials can be slowly broken down by mechanical stresses, biodegradation mediated by endogenous enzymes in the skin or host microbes. We hypothesised that the biodegradation products of hair may possess bioprotective properties, which supplement their physical protective properties. Although evolutionary processes have led to a reduction in the amount of hair on the human body, it is possible that the bioprotective properties of hair biodegradation products have persisted. The human skin is exposed to various environmental carcinogenic factors. Therefore, we hypothesised that the potential bioprotective mechanisms of hair degradation products affect melanoma growth. We used pepsin to partially digest hair enzymatically, and this process produced a water-soluble lysate containing a mixture of peptides, including fragments of keratin and keratin-associated proteins. We found out that the mixtures of soluble peptides obtained from human hair inhibited the proliferation of human melanoma cells in vitro. Moreover, the hair-derived peptide mixtures also inhibited the proliferation of B lymphoma cells and urinary bladder cancer cells. Normal human cells varied in their susceptibility to the effects of the lysate; the hair-derived peptide mixtures modulated the proliferation of normal human fibroblasts but did not inhibit the proliferation of human mesenchymal cells derived from umbilical cord stromal cells. These results suggest that hair-derived peptides may represent a new class of anti-proliferative factors derived from basically structural proteins. Identification of active regulatory compounds and recognition of the mechanism of their action might pave the way to elaboration of new anticancer drugs.

Mesenchymal cell populations: development of the induction systems for Schwann cells and neuronal cells and finding the unique stem cell population

Mesenchymal cell populations, referred to as mesenchymal stem cells or multipotent stromal cells (MSCs), which include bone marrow stromal cells (BMSCs), umbilical cord stromal cells and adipose stromal cells (ASCs), participate in tissue repair when transplanted into damaged or degenerating tissues. The trophic support and immunomodulation provided by MSCs can protect against tissue damage, and the differentiation potential of these cells may help to replace lost cells. MSCs are easily accessible and can be expanded on a large scale. In addition, BMSCs and ASCs can be harvested from the patient himself. Thus, MSCs are considered promising candidates for cell therapy. In this review, I will discuss recently discovered high-efficiency induction systems for deriving Schwann cells and neurons from MSCs. Other features of MSCs that are important for tissue repair include the self-renewing property of stem cells and their potential for differentiation. Thus, I will also discuss the stemness of MSCs and describe the discovery of a certain stem cell type among adult MSCs that can self-renew and differentiate into cells of all three germ layers. Furthermore, I will explore the prospects of using this cell population for cell therapy.

Human feeder layer system derived from umbilical cord stromal cells for human embryonic stem cells

To evaluate the potential of human umbilical cord-derived stromal cells (hUCSCs) as a human feeder for human embryonic stem cells (ESCs). Prospective study. Laboratory of Molecular Genetics and Stem Cell Differentiation, Dental Research Institute, School of Dentistry, Seoul National University. The hUCSCs were established, and human ESCs were cultured on established hUCSCs without serum. Cell-surface markers, karyotyping, and teratoma formation. Primary cultures of hUCSCs from individual umbilical cords were maintained by an established protocol. Human ESCs on hUCSC layers were successfully maintained in serum-free culture medium past passage 30. Compared with hESCs on mouse feeder cells, the hESCs on hUCSCs showed similar levels of pluripotency-related cell-surface markers, self-renewal capacity, and teratoma formation in immune-deficient mice. These ESCs cultured on hUCSCs had a normal karyotype, even after long-term culture. The hUCSCs supported self-renewal of hESCs in serum-free conditions. This culture system has the potential to facilitate the development of clinical-grade hESCs for regenerative medicine.

High Harvest Yield, High Expansion, and Phenotype Stability of CD146 Mesenchymal Stromal Cells from Whole Primitive Human Umbilical Cord Tissue

Human umbilical cord blood is an excellent primitive source of noncontroversial stem cells for treatment of hematologic disorders; meanwhile, new stem cell candidates in the umbilical cord (UC) tissue could provide therapeutic cells for nonhematologic disorders. We show novel in situ characterization to identify and localize a panel of some markers expressed by mesenchymal stromal cells (MSCs; CD44, CD105, CD73, CD90) and CD146 in the UC. We describe enzymatic isolation and purification methods of different UC cell populations that do not require manual separation of the vessels and stroma of the coiled, helical-like UC tissue. Unique quantitation of in situ cell frequency and stromal cell counts upon harvest illustrate the potential to obtain high numerical yields with these methods. UC stromal cells can differentiate to the osteogenic and chondrogenic lineages and, under specific culturing conditions, they exhibit high expandability with unique long-term stability of their phenotype. The remarkable stability of the phenotype represents a novel finding for human MSCs, from any source, and supports the use of these cells as highly accessible stromal cells for both basic studies and potentially therapeutic applications such as allogeneic clinical use for musculoskeletal disorders.

Implementation of Combined Storage of the Hematopoietic Cord Blood Cells and Fetal Mesenchymal Stromal Cells (umbilical cord stromal cells, UCSC) from the Same Donors in Warsaw Cord Blood Bank

Project design and rationale: Expanding clinical evidence suggests, that combined transplantation of allogeneic hematopoietic stem cells (HSC) and mesenchymal stromal cells (MSC) improves the results when compared to the transplantation of HSC only by the enhancement of engraftment or reduction of the frequency and gravity of graft versus host (GVH) disease. We have modified our method [1] of the processing and liquid nitrogen–storage of umbilical cord stromal cells (UCSC, the analogue of adult bone marrow MSC) for the combined collection and banking of cord blood hematopoietic stem cells (CB HSC) and UCSC obtained from the same donor for the hematopoietic transplantation purposes. Practical outcome of the project is the availability of cryopreserved combinations of cord blood samples plus the same donor-derived fetal MSC-type cells for clinical applications.Methods: Cord blood (CB) samples were collected and processed according to the routine protocol of Warsaw cord blood bank. Cord blood samples (vol.≥90 ml) were collected before or after placenta delivery, and viability and sterility-tested suspensions of mononuclear cells were volume-reduced and frozen in 10% DMSO/5% albumin using controlled-rate freezer, and stored in liquid nitrogen storage tanks. Umbilical cords, collected from the same deliveries as CB, were transported for processing in sterile balanced salt solution (PBS) supplemented with Penicillin/Streptomycin (22–25°C, time until processing ≤24h). Following mounting on the dissecting table, they were cut into 7 cm pieces, and surgically dissected in sterile conditions. After removal of cord amnion and blood vessels, Wharton jelly was cut into strips of thickness < 2 mm, sealed in 2 freezing bags in 10% DMSO/5%albumin and frozen in control-rate freezer. For experimental purposes, another strip of Wharton jelly was frozen in third bag, stored for 14 days, thawed, and the UCSC were in vitro expanded throughout consecutive 5 passages, phenotype-analysed, and tested for their ability for adipo-chondro-, and osteogenesis. Remaining fragments of umbilical cord (blood vessels, Wharton jelly pieces) were sterility-tested, and the contaminated frozen samples were eliminated at the quarantine stage. Maternal blood examination, HLA-typing, family history analysis etc. obligatory tests for CB banking were done only once, and the results were valid both for CB HSC and UCSC, reducing the labor time and costs of tests.Results: Both in cord blood samples, and umbilical cords, the frequency of bacterial/fungal infections was <1.5%, and viability of cells was>99%. The frequency of successful expansion of frozen/thawed UCSC was >90%, and average UCSC number after expansion was 2.5× 1011cells. All UCSC were plastic adherent, expressed CD34−, CD45−, Lin1−, CXCR4−, CD29+, CD73+, CD90+, CD105+ surface markers, and were able to differentiate into adipo-, chondro-, and osteogenic lineages, thus confirming their MSC characteristics. Before the full validation of the procedures, CB HSC and UCSC obtained from the same donor are stored in Warsaw cord blood bank in separate storage tanks in −170°C in liquid nitrogen vapors. If the transplantation of cord blood cells plus umbilical cord MSC-like cells will be accepted as the standard clinical protocol, the double cassette for combined CB HSC/UCSC storage will be implemented.Conclusion: The practical result of the presented study is the implementation of the new protocol allowing for availability of cord blood cells and mesenchymal stromal cells from the same donor for clinical transplantation purposes.

Human umbilical cordstromal cells derived from Wharton's jelly

：Human umbilical cordstromal cells derived from Wharton's jelly bear the potential of stem cells. They sharethe common surface markers with mesenchymal stem cells derived from bone marrow. They havea relatively higher proliferation rate and self-renewal capacity and can be successfullydifferentiated into mature neurons, cardiomyocytes, endothelial, adipocytes, chondrocytesand skeletal cells. Researches have shown their promising foregrounds in regenerativetherapeutic applications. This article will give a review about the separation andcultivation methods, in vitro differentiation, and in vitro and in vivo transplantationexperiments of the aforementioned stromal cells. Key words: Human umbilical cord; Stromal cells; Wharton's jelly

Concise Review: Human Umbilical Cord Stroma with Regard to the Source of Fetus-Derived Stem Cells

Human umbilical cord (UC) has been a tissue of increasing interest in recent years. Many groups have shown the stem cell potency of stromal cells isolated from the human UC mesenchymal tissue, namely, Wharton's jelly. Since UC is a postnatal organ discarded after birth, the collection of cells does not require an invasive procedure with ethical concerns. Stromal cells, as the dominant cells of this fetus-derived tissue, possess multipotent properties between embryonic stem cells and adult stem cells. They bear a relatively higher proliferation rate and self-renewal capacity. Although they share common surface markers with bone marrow-derived MSCs, they also express certain embryonic stem cell markers, albeit in low levels. Without any spontaneous differentiation, they can be successfully differentiated into mature adipocytes, osteoblasts, chondrocytes, skeletal myocytes, cardiomyocytes, neurons, and endothelial cells. While causing no immunorejection reaction, they effectively function in vivo as dopaminergic neurons, myocytes, and endothelial cells. Given these characteristics, particularly the plasticity and developmental flexibility, UC stromal cells are now considered an alternative source of stem cells and deserve to be examined in long-term clinical trials. This review first aims to document the published findings so far regarding the nature of human UC stroma with special emphasis on the spatial distribution and functional structure of stromal cells and matrix, which serves as a niche for residing cells, and, secondly, to assess the in vitro and in vivo experiments in which differential stem cell potencies were evaluated.