Stem Cell Research & Therapy | Full text | Umbilical cord …

Abstract Introduction

Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are self-renewing multipotent progenitors with the potential to differentiate into multiple lineages of mesoderm, in addition to generating ectodermal and endodermal lineages by crossing the germline barrier. In the present study we have investigated the ability of UCB-MSCs to generate neurons, since we were able to observe varying degrees of neuronal differentiation from a few batches of UCB-MSCs with very simple neuronal induction protocols whereas other batches required extensive exposure to combination of growth factors in a stepwise protocol. Our hypothesis was therefore that the human UCB-MSCs would contain multiple types of progenitors with varying neurogenic potential and that the ratio of the progenitors with high and low neurogenic potentials varies in different batches of UCB.

In total we collected 45 UCB samples, nine of which generated MSCs that were further expanded and characterized using immunofluorescence, fluorescence-activated cell sorting and RT-PCR analysis. The neuronal differentiation potential of the UCB-MSCs was analyzed with exposure to combination of growth factors.

We could identify two different populations of progenitors within the UCB-MSCs. One population represented progenitors with innate neurogenic potential that initially express pluripotent stem cell markers such as Oct4, Nanog, Sox2, ABCG2 and neuro-ectodermal marker nestin and are capable of expanding and differentiating into neurons with exposure to simple neuronal induction conditions. The remaining population of cells, typically expressing MSC markers, requires extensive exposure to a combination of growth factors to transdifferentiate into neurons. Interesting to note was that both of these cell populations were positive for CD29 and CD105, indicating their MSC lineage, but showed prominent difference in their neurogenic potential.

Our results suggest that the expanded UCB-derived MSCs harbor a small unique population of cells that express pluripotent stem cell markers along with MSC markers and possess an inherent neurogenic potential. These pluripotent progenitors later generate cells expressing neural progenitor markers and are responsible for the instantaneous neuronal differentiation; the ratio of these pluripotent marker expressing cells in a batch determines the innate neurogenic potential.

Umbilical cord blood (UCB) is considered one of the most abundant sources of non-embryonic stem cells [1]. The collection of mesenchymal stem cells (MSCs) from UCB that is discarded at the time of birth is an easier, less expensive and non-invasive method than collecting MSCs from bone marrow aspirates [2]. These MSCs attract special interest due to these specific advantages over embryonic and adult stem cell counterparts, since there are also no ethical issues associated with UCB. Another important characteristic of UCB-MSCs is that they are less immunogenic, and therefore do not elicit the proliferative response of allogeneic lymphocytes in vitro [3]. UCB-MSCs expanded in vitro also retain low immunogenicity and an immunomodulatory effect. Moreover, cells derived from the UCB elicit a lower incidence of graft rejection and post-transplant infections compared with other sources [4].

Considering these facts, UCB-derived MSCs can therefore be effectively utilized for therapeutic applications of various diseases. These applications include cell-based therapy to replenish degenerated neurons, cardiac cells, muscle cells, chondrocytes, and so forth. However, the potential application of these cells for various purposes requires extensive characterization, and requires standardization of reproducible differentiation protocols with ultimate functional characterization of the differentiated cells. Morphologically, the MSCs are adherent, fibroblast-like cells [5] with multipotent differentiation potential and thus can be induced to differentiate into cells of multiple lineages such as adipocytes, osteocytes, chondrocytes, myocytes, hepatocytes, neurons and astrocytes [6-10]. Several groups have explored the possibility of generating functional neurons from UCB-MSCs to use for various neurodegenerative diseases. Administration of human umbilical cord blood (hUCB)-MSCs was found to be feasible treatment for brain injuries such as stroke and other degenerative disorders [11,12]. Transplanting hUCB-MSCs in spinal cord injury animal models has shown significant improvement in neurological function [13].

Even though the hUCB-derived cells have been shown to differentiate into different lineages [14], a high potential for neuronal differentiation has been shown with extensive exposure to multiple combinations of growth factors [15-18]. In vitro treatment with -mercaptoethanol and retinoic acid resulted in a very drastic difference in cellular morphology of MSCs from fibroblastic to spindle-shaped with elongated processes resembling a neuronal phenotype [19]. Previous reports have shown that hUCB-MSCs can be transdifferentiated into neuronal lineage by treating with nerve growth factor and retinoic acid. This multi-lineage differentiation capacity, the expression of neural properties and overlapping genetic programs for hematopoiesis and neuropoiesis [20] suggest that hUCB cells may have the ability to transdifferentiate in to neural cells. Few reports have shown that UCB-derived progenitors can express Oct3/4, Sox2, Nanog and Rex1, which are pluripotent/multi-lineage markers and could possibly differentiate into multiple lineages [21-23]. Convincing evidence therefore appears to show that hUCB-MSCs can be used as a good source for generating neurons, but there exists a considerable difference in the neurogenic potential of different batches of MSCs obtained from UCB.

The multi-lineage differentiation capacity and an inherent neuronal differentiation potential observed in a few batches made us characterize the hUCB-derived cells in detail with respect to neurogenic potential, since evaluating the neurogenic potential of hUCB-MSCs isolated for any possible cell replacement therapeutic applications is really important. Our results have in fact shown that the hUCB-MSCs varied in their neurogenic differentiation potential between samples, with a few showing the presence of a unique population of cells with inherent neurogenic potential even though they phenotypically express MSC markers.

UCB samples were collected from 45 full-term delivery cases with previous consent from the mothers according to the Institute's human ethical committee guidelines. The UCB samples collected were coded as hUCB-MSC SCB1 to SCB45. The samples used for mononuclear cell (MNC) isolation alone were coded as hUCB MNC01 to MNC03. Clinical parameters for the mother and baby were also noted for latter correlation with the yield of MSCs. The parameters recorded were the gestation period, UCB blood volume collected, and body weight of the baby (see Table S1 in Additional file 1).

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