Mammalian pluripotent stem cells, methods for their production, and uses thereof
A pluripotent stem cell, mammalian technology, applied in the field of mammalian pluripotent stem cells, their production and their uses, and can solve problems such as tumorigenesis
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Embodiment 1
[0072] 8.1. Example 1: Generation of NEDAPS cells using BMP2
[0073] Materials and Methods: Ten mice were anesthetized (under an IACUC-approved protocol) and the right sciatic nerve was exposed using standard methods. In 7 animals, 50 ng of BMP2 was placed directly onto the nerve. In 3 control animals, no agent was administered to the nerve.
[0074] Animals were humanely sacrificed after 12, 24 or 48 hours, re-exposed and the sciatic nerve harvested. Nerves were fixed in formaldehyde and embedded in paraffin, and sectioned by standard methods.
[0075] Results: Untreated nerves appeared normal (see Figure 1A ), except that some mild inflammation may be found, which is thought to be due to surgical exposure.
[0076] BMP2-treated nerves were found to be fragmented and damaged (see Figure 1 ), but significant cell proliferation within the nerve was noted. Note that the treated nerves are spontaneously fragmented during and after sectioning. Nerves treated with BMP2 were...
Embodiment 2
[0077] 8.2. Example 2: Generation of NEDAPS cells using IM injected BMP2
[0078] Materials and Methods: Twenty mice were anesthetized and 50 ng or 100 ng of BMP2 were injected intradermally (IM) into the right tendon muscle of each mouse. Mice were sacrificed after 24 hours, 48 hours or 72 hours. Harvest the tendon without dissection to the sciatic nerve, but take care that the harvested tissue encompasses the usual anatomical location of this nerve. Contralateral (untreated) tendon muscle was obtained from 4 animals as control tissue.
[0079] Because BMP2 was administered by IM injection, it was difficult to know how close the injection site was to any given microscopic area after tissue harvesting and processing. Sections were stained for an array of stem cell markers and a panel of inflammatory markers.
[0080] RESULTS: Nerves from limbs injected with BMP2 were disrupted and slightly fragmented even in animals at 24 hours (see Figure 2B and 2D ). Nerves from tre...
Embodiment 3
[0082] 8.3. Example 3: Production, isolation and culture of NEDAPS cells
[0083] In vivo NEDAPS cell production: All animal activities were performed at the Wichita State University Animal Care Facility and were approved by the Wichita State University Animal Care and Use Committee. Female BALB / c mice, 8-12 weeks old, were purchased from Jackson Laboratories (Bar Harbor, ME) and allowed to acclimatize to the facility for at least 1-6 weeks prior to study use. On the day of surgery, for prophylactic analgesia, mice received 0.05 mg / kg buprenorphine by subcutaneous injection 1 h before surgery. Mice were anesthetized by intraperitoneal injection of 90 mg / kg of ketamine and 8 mg / kg of xylazine, and supplemented with 1%–2% isoflurane through a nose cone. The right leg of each animal was shaved and the surgical area was disinfected with povidone-iodine and ethanol. An incision is made on the lateral aspect of the thigh and the sciatic nerve is exposed by blunt dissection. The n...
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