Stem cell maturation method for all tissue lines
A stem cell, embryonic stem cell technology, applied in the field of cell therapy, cell biology, stem cell therapy
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[0041] Preparations of HSCs may be obtained from the same species, or they may be from different species. Transfer of HSCs can be into a host of the same species or into a host of a different species.
[0042] Alternatively, primed HSCs can be used to obtain cells for therapy in the treatment of abnormal conditions and tissue repair.
[0043] In another embodiment of the invention, the therapeutic composition comprises enucleated adult stem cells having nuclei from primordial sex cells or embryonic stem cells. Therapeutic compositions can be used to regenerate diseased or injured tissue of an animal in need thereof. Diseased or damaged tissue can include, for example, heart tissue, lung tissue, and other bodily tissue.
Embodiment 1
[0049] Example 1 Isolation of primordial sex cells (PSCs)
[0050] Anesthetize the mammal or animal, remove the gonads, and transect. Primary sex cells (PSCs) were isolated with the aid of a microscope. Alternatively, microscopically assisted isolation of PSCs can also be performed using biopsy punches from the gonads. Microscopically, PSCs have the morphology of stem cells (ie, large, round, smooth) that were mechanically retrieved from the gonads. In particular, spermatogonia and oogonia are retrieved from the gonads. In particular, type A and type B spermatogonia are retrieved.
[0051] To obtain egg(s), the animal is hyperovulated, at least one egg is retrieved, and placed alive on a nutrient medium. Using a micropipette to hold the egg in place, use another micropipette to enter the egg until the tip is adjacent to the nucleus of the egg. Enucleation of eggs can be done by applying a small amount of vacuum to a micropipette. Egg (ln) nuclei were discarded. The en...
Embodiment 2
[0053] Example 2 Isolation and Purification of Type A Spermatogonia
[0054] The following is an illustrative example for the isolation and purification of type A spermatogonia. In step 1, testes were removed from 6-day-old donor mice (n=8) and placed in petri cultures with sterile phosphate-buffered saline (PBS) containing 10% penicillin-streptomycin in the dish.
[0055] Next, in step 2, the testes are demembraned under a dissecting microscope, and the semiferous cord / tubule is collected, pooled, and placed in a conical centrifuge tube containing the following solution: Dulbecco modified A solution of 2 mg / ml collagenase (Sigma Chemicals, St. Louis, MO) and 10 μg / ml Dnase I (Sigma Chemicals, St. Louis, MO) in Eagle's medium (DMEM; Specialty Media).
[0056] In step 3, the centrifuged contents were incubated on a shaker at 37°C for 30 minutes with occasional gentle pipetting to separate the interstitial Leydig cells from the seminiferous tubules.
[0057] In step 4, afte...
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