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Transdifferentiation of Bone Marrow Mesenchymal Stem Cells into Neural Cells via Cerebrospinal Fluid

Dina Y. Otify, EmanA. Youssef, Naglaa B. Nagy, Mona K. Marei, Magda I. Youssif

Biomedicine and Biotechnology. 2014, 2(4), 66-79 doi:10.12691/bb-2-4-2
  • Figure 1. Photomicrographs showing steps of surgical isolation of BM-MSCs: (a)Rabbit femur after exposure. (b) Sample collection using a sterile syringe containing anticoagulant under aseptic condition. (c) A sterile syringe containing the harvested bone marrow sample
  • Figure 2. Photomicrographs by IPCM showing themorphological features and proliferation of cultured undifferentiated BM-MSCs: (a) Undifferentiated BM-MSCs appeared as few clusters of spindle shaped cells after 4 days of cultivation (Scale bar = 500µm).(b) Higher magnification of the white square shows spindle shaped cells at the same day (Scale bar = 100µm).(c) Colony size expanded within 14 days of cultivation, and the cells arranged in a swirling pattern (Scale bar = 500µm).(d) Highly crowded bipolar fibroblast BM-MSCs at 21 days of primary culture (Scale bar = 500µm)
  • Figure 3. Photomicrographs showing the morphological features of the undifferentiated BM-MSCs: (a)The undifferentiated BM-MSCs stained with H&E and examined with light microscope, shows static morphology as bipolar fibroblastic structure (Scale bar = 50µm).(b) The undifferentiated BM-MSCs examined with SEM shows flatten or spindle-shape cells (Scale bar = 50µm).(c) Clusters of undifferentiated BM-MSCs examined with CLSM, expressed positive CD146 immunoreactivity and exhibited as spindle or fibroblastic-like morphology (Scale bar = 100µm).(d) Higher magnification of undifferentiated BM-MSCs examined with CLSM, and having static morphology as bipolar fibroblastic structure with positive CD146 (Scale bar = 50µm)
  • Figure 4. Photomicrographs shows proliferation and differentiation of neural progenitor-like cells from BM-MSCs after incubation in culture media supplemented with CSF: (a) Phase-contrast microscopy picture showing differentiated bipolar neuronal progenitor cells (red arrows) after 4 days, yellow arrow indicate pro-oligodendrocyte precursor cell (Scale bar = 100µm). (b) Phase-contrast microscopy picture showing formation of connections between branched oligodendrocyte progenitor cell (yellow arrow) and attached with them oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells (Orang arrow) (Scale bar = 100µm). (c, c’) Phase-contrast microscopy picture showing differentiated multipolar neuronal progenitor cells (red arrow) after 4 days, orang arrows indicate multinucleated neural progenitors (stem cells) differentiated into oligodendrocyte-type-2 astrocyte (O-2A)-like progenitor cells (Scale bar = 100µm). (d, d’) Phase-contrast microscopy pictures showing neural elongated-like stem cell after 7 days (Scale bar = 100µm)
  • Figure 5. Photomicrographs showing the characterization of neural progenitor-like cells differentiated from BM-MSCs after 4 days ofincubation in culture media supplemented with CSF: (a) Differentiated neural progenitor cells stained strongly with silver impregnation stain (Scale bar = 200µm). (b, c) Higher magnification to the previous showing differentiated multipolar (red arrows) and bipolar (white arrow) neuronal progenitor cells (Scale bar = 50µm).(d) Immunofluorescence image showing differentiated neuronal progenitor cells expressed positive GFAP immunoreactivity culturing (Scale bar = 50µm).(e) Immunofluorescence image showing differentiated neuronal progenitor cells attached with pro-oligodendrocyte precursor cell expressed positive GFAP immunoreactivity (Scale bar = 50µm)
  • Figure 6. Photomicrographs by IPCM showing the morphological features of the differentiated neural-like cells: (a) Neuronal precursor cell has pyramidal soma with rounded nucleus and elongated neurite (24 hrs after induction)(Scale bar = 100µm).(b) More developed immature neuronal cells possess two to three short neurites after 3 days of inductionin CSF(Scale bar = 100µm). (c) Completely developed maturemultipolarneuron with distinct soma and distinct axon and distinguished multi-branched dendrites emanated from it after 5 days of induction.(d)Mature "bipolar" neuron developed within 9 days of induction(Scale bar = 100µm). (e) Immature Astrocyte cell has polygonal cell body with rounded nucleus and more processes emerged from it within 3 days of incubation (Scale bar = 100µm). (f) Mature astrocyte completely differentiated after 5 days of induction, with distinct star like structure soma and dense plasma to fibrous dendrites(Scale bar = 100µm)
  • Figure 7. Photomicrographs showing the proliferation and differentiation of astroglial and mature astrocytes from BM-MSCs after incubation in culture media supplemented with CSF: (a)The white circle area in the phase-contrast microscopy picture showing neural stem cells with astroglial morphology describing a neurogenic niches of highly replicating cells (i.e. small neurogenic islands where neurons and glia are continuously generated) (Scale bar = 500µm). (b) Mature star-like structure astrocyte after 5 days (Scale bar = 100µm)
  • Figure 8. Phase-contrast microscopy picture showing the proliferation and differentiation of mature neural cells from BM-MSCs after incubation in culture media supplemented with CSF:(a)Highly enriched area of differentiated mature bipolar neuronal cell (green arrow) and multipolar neuronal cell (red arrow) with multi-branched neurites contacting with mature oligodendrocyte (yellow arrow) after 5 days, white arrow indicate pro-oligodendrocyte contacting neuritis branches (Scale bar = 500µm). (b, b’) Enlargement of the red square area showing mature oligodendrocyte (yellow arrow) with granular cytoplasm which intensively produces myelin component and extending sheaths (yellow stars) contacting neurite branches (orange arrows) of the multipolar neuron (red arrow) for enveloping, black arrow showing cell body of unipolar neuron with coiled proximal axon (Scale bar = 100µm). (c)Mature multipolar neuronal cells after 5 days of culturing (Scale bar = 100µm). (d)Mature oligodendrocyte after 8 days, yellow arrows indicate dense granular cytoplasm area (rough endoplasmic reticulum during myelination), red arrow showing sheaths of membrane enveloping the contacting axon (white arrows) of the neighboring neuron (Scale bar = 100µm)
  • Figure 9. Photomicrographs shows the morphological features of the differentiated neural-like cells: (a)Neural precursor cell stained with H&E, with elongated neuriteemerged from the soma after 24hrs(Scale bar = 50µm). (b)Semi-differentiated neurons stained with H&E after 5 daysof induction,with distinct soma, axonand distinguished multi-branched dendrites emerge from it(Scale bar = 50µm).(c)Fully differentiated mature astrocytes after 5 daysof induction stained with H&E,with distinct soma and multi-branched dendrites (Scale bar = 50µm).(d) Neuronal precursor-like cell examined with SEMafter 24 hrsof induction, has pyramidal or cone-like structure soma with central rounded nucleus and extending neurite (Scale bar = 10µm). (e)Mature multipolar neuron (red arrow) after 5daysof induction, examined with SEM, has distinct soma with clearly appeared central rounded nucleus, distinct axonand multi-branched dendrites (Scale bar = 50µm). (f)After 9 days of induction,a very elongated bipolar neuron cell observed using SEM (Scale bar = 50µm)
  • Figure 10. Phase-contrast microscopy picture showing cytological characterization of mature neural-like cells differentiated from BM-MSCs after incubation in culture media supplemented with CSF using special stains: (a) Before the induction process (0 day), the cultured cells appeared normally as spindle shape with weak reaction activity and clearly stained nuclei(Scale bar = 50µm).(b) Differentiated neural-like cells stained with cresyl violet after 5 days of induction showing strong concentration of Nissl bodies "NB" granules surrounded the nucleus "N"(Scale bar = 50µm).(c) Fully differentiated mature neuronal-like cells after 9 days of induction, having soma counterstained with cresyl violet (red arrow)and multi-branched neurits impregnated with black silver stain (blue arrow), forming fibrous network-like structure (red square)(Scale bar = 200µm).(d, e)Higher magnification of the neuron-like cells and fibrous neural-like network in the previous figure (Scale bar = 50μm).(f)Fully differentiated mature neuronal-like cells after 14 days, forming fibrous network-like network (red square) (Scale bar = 200µm).(g)Very dense fiberousneuronal network shown by SEM (Scale bar = 5μm).(h) Undifferentiated BM-MSCs before the induction process (0 day), showing spindle shaped cells with very weak PASstain in their cytoplasm(Scale bar = 50µm).(i, j)Mature multipolar neuronal-like cell stained with PAS showing strong glycogen activity, and stronglystained withcresyl violet, respectively(Scale bar = 50µm).(k)Mature bipolar neuron performed after 7 daysof induction which possess distinct soma with strong glycogen content(Scale bar = 50µm).(l) Mature astrocyte-like cells stained with PAS after 5 days of induction in CSF, revealedintense glycogen content(Scale bar = 50µm)
  • Figure 11. characterization of mature neural-like cells differentiated from BM-MSCs after incubation in culture media supplemented with CSF using GFAP Immunolabelinginvestigated by CLSM: (a)Negative GFAP expression in the undifferentiated BM-MSCs before the differentiation process (0 day)(Scale bar = 50µm).(b)Immunofluorescence image showing positive GFAP immunolabeling for astrocytes within 3 days of incubation,investigated by CLSM(Scale bar = 50µm).(c)Strong GFAP immunolabeling for pseudo-unipolar neuronal-like cell after 5 days, investigated by CLSM(Scale bar = 50µm).(d, d')Mature unipolar neuronal-like cell showing positive GFAP immunoreactive and silver stain, respectively (Scale bar = 50µm).(e, f) Immunofluorescence image showing mature neuronal and oligodendrocyte cells GFAP immunoreactive (Scale bar = 50µm). (e’, f’) Inverted phase micrograph of e) and f) showing perineural oligodendrocytes; Oligodendrocyte (red arrow), Neuronal cells (yellow arrow). (Scale bar = 50µm)