Long-term cultured human umbilical cord neural-like cells transplanted into the striatum of NOD SCID mice

Abstract

The use of stem cells and other cells as therapies is still in its infancy. One major setback is the limited survival of the grafts, possibly due to immune rejection. Studies were therefore performed with human umbilical cord blood cells (HUCB) to determine the ability of these cells to survive in vivo and the effect of the immune response on their survival by transplantation into the normal striatum of immunodeficient NOD SCID mice. Long-term culture of HUCB cells resulted in several different populations of cells, including one that possessed fine processes and cell bodies that resembled neurons. Their neuronal phenotype was confirmed by immunohistochemical staining for the early neuronal marker TuJ1 and the potentially neural marker Nestin. Five days after cell transplantation of this neuronal phenotype, immunohistochemical staining for human mitochondria confirmed the presence of living HUCB cells in the mouse striatum, with cells localized at the site of injection, expressing early neural and neuronal markers (Nestin and TuJ1) as well as exhibiting neuronal morphology. However, no evidence of surviving cells was apparent 1 month postgrafting. The absence of signs of T cell-mediated rejection, such as CD4 and CD8 lymphocytes and minimal changes in microglia and astrocytes, suggest that cell loss was not due to a T cell-mediated immune response. In conclusion HUCB cells can survive long-term in vitro and undergo neuron-like differentiation. In mice, these cells do not survive a month. This may relate to the differentiated state of the cells transplanted into the unlesioned striatum, rather than T cell-mediated immunological rejection.

Fig. 1

The morphological comparison of HUCB cells between short- and long-term culture. The bright field micrographs showed that (A) HUCB mononuclear cells formed clusters 1 DIV after culture. (B) HUCB cells morphologically presented high heterogeneity on day 20 in culture, bigger flat round cells adhered to the surface of vessels (asterisks) with small round and differentiated cells sitting on top of the adherent cells (arrows). (C) and (D) show the morphology of long-term cultured HUCB cells. (C) These cells grew well and were able to form clusters. (D) Differentiating cells had long single or multiple processes (arrows). Scale bar: 20 μm (A and C) and 40 μm (B and D).

Fig. 2

Immunofluorescence microphotographs showing different incidences of the expression of hematopoietic and early neuronal antigens in short- and longer-term cultured HUCB cells. (A) More then 90% of cells expressed CD45 on 5-day culture (green) and a low number of CD45 positive cells were present in 192-day culture (D, green). (B) A small amount of cells that scattered in the short-term culture (5 DIV) presented Nestin staining (green). However, numerous cells in longer-term culture (143 DIV) expressed Nestin antigen (E, green). Most Nestin positive cells were clustered and resembled a neurosphere. (C) A few cells at day 15 were TuJ1 positive (red) and 60-80% of cells at day 143 exhibited TuJ1 positivity (red). Blue DAPI counterstaining was used for clear identification of all cultured cells in (D-F). Scale bar: 20 μm (A), 40 μm (B and C) and 50 μm (D-F).

Fig. 3

Immunofluorescence microphotographs showing the pattern of human cell markers at the injection site for HUCB cells in mouse striatum, 5 days after grafting. (A) Human mitochondria (HuMi; green) reveals the presence of transplanted cells amongst a background of non-staining mouse cells. Note the variety of cell shapes, with some possessing long processes. (B) HuMi staining (green) is shown against a background of TuJ1 (red) endogenous neuronal staining. (C) The human neural-specific Nestin stain (green) demonstrates the presence of HUCB cells at the injection site in the mouse striatum. In B and C nuclei are visualized with DAPI staining (blue). A subpopulation of the grafted HUCB are also TuJ1 positive (white/yellow in appearance), suggesting differentiation into neuron-like cells. Notice the lack of migration of these cells away from the injection site. Scale bar = 100 μm in all figures.

Fig. 4

Immunofluorescence microphotograph depicting the presence of microglia at the injection site for HUCB cells in mouse striatum, 5 days after grafting. CD11b staining (red) was used to demonstrate the increased presence of microglia and macrophages at the injection site of the HUCB cells. The morphology of the stained cells - typically branched - reveals a general lack of activated cells (shown by unbranched, rounded cells) of these cells at 5 days postgrafting, demonstrating the absence of a fully developed immune response. Scale bar = 200 μm.

Fig. 5

Immunofluorescence microphotograph demonstrating the incorporation of the transplanted HUCB cells into the striatum at the injection site. The HUCB cells, stained by Nestin (green) are surrounded by fibronectin (red). Fibronectin is an important substrate for axonal sprouting and guidance and its colocalisation (yellow) with the HUCB cells suggests their attempted integration into the striatum. DAPI staining (blue) reveals the presence of all endogenous (and non-neuronal HUCB) cells within the injection site.