The standard ethical guidelines were followed. First, the human umbilical cords were obtained just before disposal from full-term births from local obstetricians after informed consent. Then, the umbilical cord was washed several times with PBS to flush out the blood followed by removal of the umbilical blood vessels. The remaining tissue was cut into small pieces. The explants (~ 2- to 5-cm lengths) were placed in 60 mm culture dishes and cultured in DMEM medium with high glucose (Hyclone) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), penicillin G (100 units/ml), streptomycin (100 µg/ml), and amphotericin B (25 mg/ml).²⁴ The cells were incubated at 37°C in the incubator with 5% CO2 and 95% humidity. The plates were left undisturbed for 5 to 7 days and thereafter medium was changed every 3^rd day. The cells begin to appear in 8 to 10 days of culture. When cells reached 70% to 80% confluency, the cells were detached with 0.25% trypsin-EDTA (Hyclone) and passaged subsequently.

The cells were taken and fixed with acetone (for c-KIT) at -20^oC or 4% paraformaldehyde (for OCT-4) at room temperature for 15 min. The immunocytochemistry was carried out as described in Rajan et al.²⁵ Primary antibodies concentrations used were 1:10 for c-KIT and OCT-4 (Cell Signalling). Secondary antibody (Jackson Immuno Research Laboratories) was used at 1:500 dilution. All cultures were counterstained with 1mg/ml Hoechst 33342 (Sigma-Aldrich) to visualize the nucleus in individual cells.

Around 70% confluent HUMS cells were harvested and stained with the fluorophore- tagged antibodies against CD markers following the provided protocols (Serotec). The quantitation of the TFs was carried out using the standard ELISA kits (R & D Systems).

Total RNA was extracted from umbilical cord MS cell cultures (P5-8) or the NSC34 motor neurons (27) using TRI reagent (Sigma). RNA was then reverse transcribed (RT) with MMLV reverse transcriptase using random hexamers in the presence of RNAse inhibitor. Primers used are given in (Table 1). PCR was carried out using Veriti Thermal Cycler (Applied Biosystems). The amplicons were then separated by agarose gel electrophoresis (1–1.8%).

HUMS cell cultures at 70% confluence were maintained in DMEM medium with high glucose (Hyclone) supplemented with 10% fetal bovine serum (FBS), penicillin G (100 units/ml), streptomycin (100 µg/ml), and amphotericin B (25mg/ml) at 37°C for 48 hours. The medium of the HUMS cells referred to as conditioned medium (CM) from these cultures was collected, filtered through 0.2mm filter (Millipore) and used for neurite outgrowth assay.

NSC34, a mouse spinal cord motor neuron cell line²⁶ was plated at a density 1 × 10³ cells/ml in 35mm culture plate(s). After 24 hours, the medium was replaced with CM. Control NSC34 cells were maintained in normal growth medium (DMEM supplemented with 10% FBS) under the same conditions. Neurite outgrowth was measured after 48 and 96 hours.

Identification of the differentiated & undifferentiated NSC34 cells was done on the basis of their characteristic size and distinctive morphology. The undifferentiated cells are smaller in size and generally circular in shape while the differentiated cells are bigger with neuronal morphology and having extensive neurites. Approximately 100–150 cells/field were quantified in each set of experiments and the fraction of differentiated cells was determined after 48 hours and 96 hours.

NSC34 cells were viewed using phase contrast microscopy (OlympusI×71). Images were acquired using CCD camera (Jenoptik) and analysed using ImageProPlus software. Images were taken of 5 non-overlapping visual fields (using 10 × objective) for each culture condition and in 3 independent experiments. Neurite lengths of every NSC34 cell (~100–150 cells/field) within each field of view were measured by tracing the lengths of the neurites using the measurement tool of ImageProPlus software.

Cell viability was determined by the standard MTT assay. Briefly, the cells were washed with PBS twice, followed by the addition of 1ml of 5mg/ml MTT in PBS and incubated at 37^oC for 3 hrs. This solution was removed and 1 ml of formazan solubilizing solution was added, kept for 15 min., solubilised and O.D. was measured at 570 nm.

Western blotting was carried out as described in Rajan et al.²⁵

Mean, standard deviation and P values were calculated using the Statistical Analysis software Sigma Plot 10.0. In all the graphs the error bars represent standard deviation. The P values were determined using t-test.

When the human umbilical cord Wharton jelly devoid of the blood and blood vessels were placed in the regular culture medium in vitro, colonies started growing from them (Fig. 1A-a). From these, individual cells could be obtained (Fig. 1A-b). These cells, when passaged, became the source of the HUMS cells. The HUMS cells could be maintained in in vitro cultures for more than 4 months. These were mixed population of cells. The HUMS cells were positive for several pluripotency markers like OCT-4 (Fig. 1B- and b) & c-KIT (Fig.1 B- c & d), nanog, sox -2 and alkaline phosphatase (Fig. 1C). These cells were CD44⁺ CD73⁺ CD90 ⁺ CD105 ⁺ CD34⁺ and HLA-DR^- (Fig. 1 D).

The MSCs are known to home in the injured site and provide protection through paracrine factors and immune modulation. Hence, we measured the neurotrophic factors secreted by the HUMS cells. First, we carried out the expression analysis (Fig. 2A), quantitated the content and determined their functionality (Fig. 2B and C).

The HUMSs were expressing an array of trophic factors. They were expressing the neurotrophin family of trophic factors, NGF, BDNF and NT-3 (Fig. 2A) as determined by the mRNA expression RT-PCR. Interestingly, all the three neurotrophic factors are expressed in the HUMS cells. We further analysed the expression of other trophic factors which act as neurotrophic factors for neurons, namely, GDNF, IGF-1, VEGF and CNTF. Of these, except CNTF the other three TFs were expressed (Fig. 2A- RT-PCR). Thus, the HUMS cells were expressing several trophic factors. Of these, the neurotrophin family TFs, BDNF, GDNF and NT-3 are expressed at high levels (Table 2). The rest of the three trophic factor levels were below the detection of spectrophotometric ELISA method. Then we addressed whether these trophic factors are functional.

Functionality of the neurotrophic factors secreted by the HUMS cells

HUMS cells conditioned medium (CM) induce differentiation in the motor neuron cell line, NSC34.

The well established mouse spinal cord motor neuron cell line, NSC34 mostly remain undifferentiated under normal conditions. The undifferentiated cells are smaller, generally grow in clusters or aggregates and have rounded morphology (Fig. 2B-a). After treatment with CM, there was significantly higher number of differentiated cells (Fig. 2B-b and 2C), which increased with time (Fig. 2C). Differentiated NSC34 cells exhibited typical neuronal morphology (Fig. 2B-b), were bigger in size and had long characteristic neurites. A proportional increase in the number of differentiated cells (Fig. 2C) clearly seen after 48 hours and 96 hours of CM treatment further validates the functionality of trophic factors present in the conditioned medium.

Generally, neurotrophic factors induce differentiation and neurite outgrowth in the neuronal cells. The secreted-TFs from the HUMS cells, present in the CM robustly induced differentiation, neurite extension (Fig. 2B-b, arrows) and more importantly networking (shown in the black square Fig 2B-b) in the mouse spinal cord motor neuron cell line, NSC34 (Fig 2B-b, D). The neurites’ extension increased with longer incubation with the CM. Around 0.6 mm long neurite extension could be achieved when the NSC34 motor neurons were treated with CM for 6 days. Thus, proving that the TFs expressed in the HUMS cells are functional and they can induce differentiation and neurite outgrowth (arrows) (Fig. 2B-D). Moreover, only when we inhibited all the 5 TFs with their respective antibodies simultaneously, the neurite extension was abolished (Fig. 3B) to the control level indicating several backup mechanisms for neurite extension in the motor neurons.

In order to determine that the trophic factors and their signaling contributed to neurite extension, we determined the expression of receptors for the TFs that were found to be secreted by HUMS cells. Except for the BDNF receptor, Trk B, the motor neuron cell line expressed the receptors for NGF- TrkA; NT-3- TrkC; GDNF- GDNFRa1, a2 and a3; VEGF receptor–Flk-1 or VEGFR2; IGF1 receptor -IGF1R as determined by RT-PCR (Fig. 3A). Further, to reinforce that the TFs are indeed functional and acting through their tyrosine kinase pathway, we treated the motor neuron cells with the TrK inhibitor, K252a. K252a had the effect in a narrow window of 20 nM. At 2 nM the motor neurons cells were healthy with long processes (data not shown) while 20 nM shortened the processes drastically. The higher concentration of 200 nM became toxic to the cells with a flattened and bloated appearance with thin neurites in few cells. The reduction in neurites with K252a provides the evidence that TFs are acting through the TrKs.

Though the TFs use multiple pathways to bring about neuronal differentiation and neurite outgrowth, the pathway utilized will depend on the cell type. One of the important pathways is through PI3K-Akt. When the motor neurons cells in the CM was treated with the PI3K inhibitor Ly294002, the neurite extension and differentiation was completely abolished at the routinely used concentration of 50 mM (Fig. 4B-c). Hence, PI3K –Akt pathway is the major player in the motor neuron differentiation and neurite extension. Additionally, the motor neurons utilize the MAPK pathway of RAS- ErK1/2 as ErK1/2 is strongly phosphorylated (~30% increase) upon CM treatment (Fig. 5 A & B). Thus, these two pathways seem to be the major pathways in the motor neurons for differentiation and neurite extension.

To further understand the signaling cascades activated by the TFs in the motor neurons, we treated the motor neurons with and without CM with cAMP pathway activators forskolin (adenylate cyclase activator), Dibutyryl cAMP (dbcAMP – analog of cAMP). Both these treatments did not induce any differentiation of neurite extension in the motor neurons without CM. No increase in the differentiation, neurite extension and networking was noticed with these cAMP activators in the presence of the cAMP activators. But the motor neurons do require optimal cAMP signaling as these cells were dead in the presence of higher concentration of forskolin or H89 the Protein kinase A inhibitor. Similarly, treatment with the inhibitors of the cAMP pathway, SQ22536 and Rp-cAMP did not have any effect on the motor neurons with and without CM (Fig. 6). Thus, the motor neurons do not require the cAMP cascade for differentiation and networking in the presence of the CM.

Despite generally known as the specific cAMP pathway Protein kinase A inhibitor, H89, completely inhibits MSK1, ROCKII and RSK or S6K in in vitro kinase assay. In order to inhibit PKA, we treated the motor neuron cells with H89. This led to almost complete death while motor neurons treated with CM did not show cell death (Fig. 7A and B). Intriguingly a pathway switch is happening for cell survival itself, when the TFs in the CM are inducing differentiation and neurite extension in the motor neurons. Moreover, H89 induces more extensive networking in the CM treated motor neurons suggesting that a combination of HUMS. cells and H89 could be tried for the protection in the ALS model mice/rats.