Preparing a population of human late stage motor neuron progenitor cells
Motor neuron progenitor (MNP) cells and populations of MNP cells, are provided, in particular, populations of human late stage MNP cells having a purity of greater than about 65% late stage MNP cells and high-purity populations of MNP cells having greater than 95% viable cells, as well as method of making and using the same, including deriving late stage MNP cells from pluripotent embryonic stem cells, producing high-purity populations of late stage MNP cells, producing populations of viable MNP cells, transporting viable MNP cells, and transplanting MNP cells.
1. A method of preparing a population of viable late stage human motor neuron progenitor cells comprising:
(a) obtaining population of human late stage motor neuron progenitor cells having a purity of greater than about 65% late stage motor neuron progenitor cells, wherein the population is cultured on an adherent substrate and provides a layer of motor neuron progenitor cells;
(b) dissociating the layer of motor neuron progenitor cells cultured on the adherent substrate to provide a cell suspension;
(c) allowing the cell suspension to rest in a non-adherent environment for a period of time sufficient for floating microspheres comprising viable cells to form;
(d) selecting the floating microspheres comprising viable cells; and
(e) collecting the microspheres to provide a population of viable late stage human motor neuron progenitor cells, wherein the purity of the cells is greater in step e than in step a,
further comprising enzymatically dissociating the layer of motor neuron progenitor cells, and washing the cell suspension after dissociating.
2. A method of preparing a population of viable human late stage motor neuron progenitor cells comprising:
(a) obtaining population of human late stage motor neuron progenitor cells having a purity of greater than about 65% late stage motor neuron progenitor cells, wherein the population is cultured on an adherent substrate and provides a layer of motor neuron progenitor cells;
(b) dissociating the layer of motor neuron progenitor cells cultured on the adherent substrate to provide a cell suspension;
(c) allowing the cell suspension to rest in a non-adherent environment for a period of time sufficient for floating microspheres comprising viable cells to form;
(d) selecting the floating microspheres comprising viable cells; and
(e) collecting the microspheres to provide a population of viable human late stage motor neuron progenitor cells, wherein the purity of the cells is greater in step e than in step a,
wherein the cell suspension is allowed to rest for between at 6 hours and about 20 hours.
3. A method of preparing a population of viable human late stage motor neuron progenitor cells comprising:
(a) obtaining population of human late stage motor neuron progenitor cells having a purity of greater than about 65% late stage motor neuron progenitor cells, wherein the population is cultured on an adherent substrate and provides a layer of motor neuron progenitor cells;
(b) dissociating the layer of motor neuron progenitor cells cultured on the adherent substrate to provide a cell suspension;
(c) allowing the cell suspension to rest in a non-adherent environment for a period of time sufficient for floating microspheres comprising viable cells to form;
(d) selecting the floating microspheres comprising viable cells; and
(e) collecting the microspheres to provide a population of viable human late stage motor neuron progenitor cells, wherein the purity of the cells is greater in step e than in step a,
further comprising selecting the floating microspheres comprising viable cells by gravitational sedimentation of the cell suspension comprising floating microspheres, wherein the floating microspheres comprising viable cells remain in the supernatant and the nonviable cells form a pellet.
4. A method of preparing a population of viable human late stage motor neuron progenitor cells comprising:
(a) obtaining population of human late stage motor neuron progenitor cells having a purity of greater than about 65% late stage motor neuron progenitor cells, wherein the population is cultured on an adherent substrate and provides a layer of motor neuron progenitor cells;
(b) dissociating the layer of motor neuron progenitor cells cultured on the adherent substrate to provide a cell suspension;
(c) allowing the cell suspension to rest in a non-adherent environment for a period of time sufficient for floating microspheres comprising viable cells to form;
(d) selecting the floating microspheres comprising viable cells; and
(e) collecting the microspheres to provide a population of viable human late stage motor neuron progenitor cells, wherein the purity of the cells is greater in step e than in step a,
further comprising selecting the floating microspheres comprising viable cells by gravitational sedimentation of the cell suspension comprising floating microspheres, wherein the floating microspheres comprising viable cells remain in the supernatant and the nonviable cells form a pellet, and additionally comprising collecting the microspheres to provide a population of viable human motor neuron progenitor cells by collecting the supernatant.
5. A method of preparing a population of viable human late stage motor neuron progenitor cells comprising:
(a) obtaining population of human late stage motor neuron progenitor cells having a purity of greater than about 65% late stage motor neuron progenitor cells, wherein the population is cultured on an adherent substrate and provides a layer of motor neuron progenitor cells;
(b) dissociating the layer of motor neuron progenitor cells cultured on the adherent substrate to provide a cell suspension;
(c) allowing the cell suspension to rest in a non-adherent environment for a period of time sufficient for floating microspheres comprising viable cells to form;
(d) selecting the floating microspheres comprising viable cells; and
(e) collecting the microspheres to provide a population of viable human late stage motor neuron progenitor cells, wherein the purity of the cells is greater in step e than in step a,
further comprising selecting the floating microspheres comprising viable cells by gravitational sedimentation of the cell suspension comprising floating microspheres, wherein the floating microspheres comprising viable cells remain in the supernatant and the nonviable cells form a pellet, additionally comprising a plurality of selecting steps, wherein the first selecting step comprises gravitational sedimentation of the cell suspension followed by collecting the supernatant of the sedimentation is repeated one or multiple times, a second selecting step comprises sedimentation of the supernatant collected from the first sedimentation followed by collecting the supernatant of the second sedimentation, and wherein optional subsequent selecting steps comprise sedimentation of the supernatant collected from the previous selecting step followed by collecting the supernatant.
6. A method of preparing a population of viable human late stage motor neuron progenitor cells comprising:
(a) obtaining population of human late stage motor neuron progenitor cells having a purity of greater than about 65% late stage motor neuron progenitor cells, wherein the population is cultured on an adherent substrate and provides a layer of motor neuron progenitor cells;
(b) dissociating the layer of motor neuron progenitor cells cultured on the adherent substrate to provide a cell suspension;
(c) allowing the cell suspension to rest in a non-adherent environment for a period of time sufficient for floating microspheres comprising viable cells to form;
(d) selecting the floating microspheres comprising viable cells; and
(e) collecting the microspheres to provide a population of viable human late stage motor neuron progenitor cells, wherein the purity of the cells is greater in step e than in step a,
further comprising selecting the floating microspheres comprising viable cells by gravitational sedimentation of the cell suspension comprising floating microspheres, wherein the floating microspheres comprising viable cells remain in the supernatant and the nonviable cells form a pellet, additionally comprising a plurality of selecting steps, wherein the first selecting step comprises gravitational sedimentation of the cell suspension followed by collecting the supernatant of the sedimentation is repeated one or multiple times, a second selecting step comprises sedimentation of the supernatant collected from the first sedimentation followed by collecting the supernatant of the second sedimentation, and wherein optional subsequent selecting steps comprise sedimentation of the supernatant collected from the previous selecting step followed by collecting the supernatant, the method comprising three selecting steps.
7. A method of preparing a population of viable human late stage motor neuron progenitor cells comprising:
(a) obtaining population of human late stage motor neuron progenitor cells having a purity of greater than about 65% late stage motor neuron progenitor cells, wherein the population is cultured on an adherent substrate and provides a layer of motor neuron progenitor cells;
(b) dissociating the layer of motor neuron progenitor cells cultured on the adherent substrate to provide a cell suspension;
(c) allowing the cell suspension to rest in a non-adherent environment for a period of time sufficient for floating microspheres comprising viable cells to form;
(d) selecting the floating microspheres comprising viable cells; and
(e) collecting the microspheres to provide a population of viable human late stage motor neuron progenitor cells, wherein the purity of the cells is greater in step e than in step a,
further comprising determining the number of viable and nonviable cells present per unit volume of solution after at least one step.
8. A method of preparing a population of viable human late stage motor neuron progenitor cells comprising:
(a) obtaining population of human late stage motor neuron progenitor cells having a purity of greater than about 65% late stage motor neuron progenitor cells, wherein the population is cultured on an adherent substrate and provides a layer of motor neuron progenitor cells;
(b) dissociating the layer of motor neuron progenitor cells cultured on the adherent substrate to provide a cell suspension;
(c) allowing the cell suspension to rest in a non-adherent environment for a period of time sufficient for floating microspheres comprising viable cells to form;
(d) selecting the floating microspheres comprising viable cells; and
(e) collecting the microspheres to provide a population of viable human late stage motor neuron progenitor cells wherein population of viable human motor neuron progenitor cells comprises greater than 95% viable cells, and wherein the purity of the cells is greater in step e than in step a.
9. A method of preparing a population of viable human late stage motor neuron progenitor cells comprising:
(a) obtaining population of human late stage motor neuron progenitor cells having a purity of greater than about 65% late stage motor neuron progenitor cells, wherein the population is cultured on an adherent substrate and provides a layer of motor neuron progenitor cells;
(b) dissociating the layer of motor neuron progenitor cells cultured on the adherent substrate to provide a cell suspension;
(c) allowing the cell suspension to rest in a non-adherent environment for a period of time sufficient for floating microspheres comprising viable cells to form; and
(d) collecting the microspheres to provide a population of viable human late stage motor neuron progenitor cells, the population of viable motor neuron progenitor cells prepared for transplantation into a subject, wherein the purity of the cells is greater in step d than in step a,
further comprising,
(e) determining the number of cells to be transplanted;
(f) determine the number of viable motor neuron progenitor cells in the collected microspheres;
(g) determining the total volume to be transplanted;
(h) mixing the number cells to be transplanted with a volume of transplant vehicle needed to achieve the cell suspension to be transplanted.