December 14th, 2017

1. What to Know Before Getting Stem Cells Dr. John Hughes, DO December 14th, 2017

2. Outline of Lecture Embryonic vs. Adult Multipotent vs. Pluripotent Mesenchymal vs. Peripheral Blood-Based Making a Stem Cell Decision

3. What are Stem Cells? • Undifferentiated biological cells that differentiate into specialized cells or divide to produce more stem cells • Have the ability to divide and generate all cell types of the organ from which they originate • Stem cell therapy can stimulate tissue re-growth and greater blood flow to the affected areas • Can be obtained from bone marrow, adipose tissue, blood, or embryonic tissue

4. Embryonic Stem Cells • Derived from the fetus • Require special regulatory approval • Mostly used for research purposes • Not readily available • Expensive • Not autologous • Ethically controversial

5. Adult Stem Cells • Derived from bone, adipose, or blood • Require physician expertise and quality control • Mostly used for regenerative and cosmetic purposes • Readily available • Less expensive • Autologous use is permitted in US (with restrictions)

6. Multipotent vs. Pluripotent Stem Cells

7. Multipotent Stem Cells • Derived from cord blood, blood, bone marrow, fat and muscle • Forms only cell types from the mesoderm • Has a development trajectory towards a specific type of cell • “Teenage cell” already differentiated into it’s target adult cell type

8. Pluripotent Stem Cells • Derived from the embryo or blood • Forms all cell types in the body except the embryo or the placenta • Does not have a specialized trajectory of development • “Young, baby cell” with great ability to differentiate into other cell types

9. Mesenchymal vs. Peripheral Blood-Based Stem Cells (Multipotent) (Pluripotent)

10. Mesenchymal Stem Cells Characteristics: • Primarily isolated from adult fat or bone marrow, or umbilical cord blood • Modulate endogenous tissue and immune cells • Have already partially differentiated • On a development trajectory towards specific target tissues • Considered multipotent: specialization potential limited to one or more cell lines • Provides over 480 growth factors, reduction in inflammation, and immune modulation that may support joint health (Multipotent)

11. Mesenchymal Stem Cells Clinical Indications / Side Effects: • Most effective clinical use: same tissues transplantation (bone marrow to bone marrow, fat to fat) • Alternative clinical use: joint (if related to autoimmune or systemic inflammation) and autoimmune disorders • These cells do not develop into new cartilage cells • Therapeutic effects are short-lived • “Recent studies have suggested that less than 1% of systemically administered MSCs persist for longer than a week following injection” (Parekkadan & Milwid, 2010, pg 2). (Multipotent)

12. Mesenchymal Stem Cells Dangers: • Harvesting of bone marrow and fat can be unpleasant • Repeat harvesting is limited • Immunomodulatory effects can predispose the patients to more infections or even cancer (Sundin et al., 2006) • Reduce inflammation for 6 months – 2 years but have limited regenerative benefits • Increased FDA restriction for non-homologous tissue use (Multipotent)

13. Peripheral Blood-Based Stem Cells Characteristics: • Originate in bone marrow and present in peripheral blood • Were thought only to exist in embryonic stem cells until Dr. Young’s discovery of them in the peripheral blood in the late 20th century (Young & Black, 2004) • Forms cells from the three primary germ-layer lineages • Lineage-uncommitted cells • Also known as very small embryonic-like stem cells (VSELs) or blastomere-like stem cells • Have a long lifespan (can double more than 70 times) • *Not derived from umbilical cord blood (mesenchymal) (Pluripotent / Embryonic-Like)

14. Peripheral Blood-Based Stem Cells (Pluripotent / Embryonic-Like) • Understanding lineage uncommitted pluripotent stem cells requires an understanding of the germ layers • Lineage uncommitted pluripotent stem cells can produce all types of cells in the germ later (Young & Black, 2004)

15. Peripheral Blood-Based Stem Cells Clinical indications: • Regenerative in their applications unlike mesenchymal • Actually develop into new target tissue such as organs, cartilage, neurons, muscle, skin, etc. • Conditions treated: traumatic brain injury, chronic pain, ligament / tendon injuries, diabetes, osteoarthritis, osteoporosis, Alzheimer’s disease, fertility, aging, etc. (Pluripotent / Embryonic-Like)

16. Peripheral Blood-Based Stem Cells Displaced (5mm) C-7 proximal spinal fracture failed to heal 9 months post trauma Pre-Treatment Post-Treatment 4 months post-treatment of peripheral blood- based stem cells – the fracture is fully healed

17. Clinical Indications • Degenerative diseases: • Diabetes • Osteoarthritis / osteoporosis • Alzheimer’s disease • Regenerative applications: • Traumatic brain injury • Joint / ligament repair • Anti-aging • Post cancer treatment • Fertility Blood-Based / Pluripotent • Tissue Replacement (Homologous Only): • Bone marrow transplant • Breast, lips, cheeks, eyes, buttocks • Systemic inflammatory conditions: • Autoimmune disorders • Acute renal failure • Myocardial infarction • Type I diabetes • Graft-vs-host disease • Systemic lupus • Pulmonary fibrosis Mesenchymal / Multipotent

18. Making a Stem Cell Decision Bone Marrow Cost: $10,000 – $300,000 Recovery time: One Month Adipose (Fat) Cost: $7,000 – $15,000 Recovery time: One Month Peripheral Blood-Based Cost: $7,500 Recovery time: Less than a week

19. Q & A Aspen Integrative Medicine 970-927-0308 aspenintegrativemedicine.com TBI Therapy 303-447-1257 tbitherapy.com aspenintegrativemedicine.com/what-to-know-stem-cells

20. References Cell Applications. https://www.cellapplications.com/stem-0 Cellular Differentiation. http://oerpub.github.io/epubjs-demo-book/content/m46036.xhtml Kunter, U., Rong, S., Boor, P., Eitner, F., Müller-Newen, G., Djuric, Z., … & Milovanceva- Popovska, M. (2007). Mesenchymal stem cells prevent progressive experimental renal failure but maldifferentiate into glomerular adipocytes. Journal of the American Society of Nephrology, 18(6), 1754-1764. MacCord, K. (2012). Mesenchyme. The Embryo Project Encyclopedia. Retrieved from https://embryo.asu.edu/pages/mesenchyme Mesenchymal Stem Cell Reagents. Retrieved from http://www.sigmaaldrich.com/life- science/cell-biology/cell-biology-products.html?TablePage=22692887 Murnaghan, I. (2016). Multipotent stem cells. Explore Stem Cells. Retrieved from http://www.explorestemcells.co.uk/multipotentstemcells.html Parekkadan B, Milwid JM. Mesenchymal Stem Cells as Therapeutics. Annual review of biomedical engineering. 2010;12:87-117. doi:10.1146/annurev-bioeng-070909-105309. Stout, C. L., Ashley, D. W., Morgan, J. H., Long, G. F., Collins, J. A., Limnios, J. I., … & Young, H. E. (2007). Primitive stem cells residing in the skeletal muscle of adult pigs are mobilized into the peripheral blood after trauma. The American Surgeon, 73(11), 1106-1110. Sundin, M., Örvell, C., Rasmusson, I., Sundberg, B., Ringden, O., & Le Blanc, K. (2006). Mesenchymal stem cells are susceptible to human herpesviruses, but viral DNA cannot be detected in the healthy seropositive individual. Bone marrow transplantation, 37(11), 1051- 1059. Tithon Biotech (n.d.). Minutevideo retrieved from http://mv.pac.io/post/55b2e2b41e1818650cc7b872 Tithon Human Sciences (2015). Peripheral blood derived pluripotent stem cell technology. Rhttps://aspenintegrativemedicine.com/wp-content/uploads/Tithon-Human-Sciences-Ortho- Case-Study.pdf Young, H. E., Duplaa, C., Romero-Ramos, M., Chesselet, M. F., Vourc’h, P., Yost, M. J., … & Tamura-Ninomiya, S. (2004). Adult reserve stem cells and their potential for tissue engineering. Cell biochemistry and biophysics, 40(1), 1-80. Young, H. E., & Black, A. C. (2004). Adult stem cells. The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology, 276(1), 75-102.

July 6th, 2017

Disclaimer:
I have no material interest or investment in any stem cell companies or products.

• I. Introduction to stem cells
• II. Mesenchymal stem cells
• III. Pluripotent stem cells
• IV. Making a stem cell decision

Embryonic Stem Cells

• Derived from the fetus
• Requires special regulatory approval
• Mostly used for research purposes
• Not readily available
• Expensive
• Not autologous

Adult Stem Cells

• Derived from bone, adipose, or blood
• Requires physician expertise and quality control
• Mostly used for regenerative and cosmetic purposes
• Readily available
• Less expensive
• Autologous use is permitted in US

Totipotent Stem Cells

• Derived from the embryo
• Forms all cell types in the body
• Used for early embryonic development
• “Baby cells” (from a zygote) that becomes a pluripotent cell

Pluripotent Stem Cells

• Derived from the embryo or blood
• Forms all cell types in the body except the embryo or the placenta
• Does not have a specialized trajectory of development
• “Youthful cell” with great ability to differentiate into other cell types

Multipotent Stem Cells

• Derived from cord blood, blood, bone marrow, fat and muscle
• Forms only cell types from the mesoderm
• Has a development trajectory towards a specific type of cell
• “Teenage cell” already differentiated into it’s target adult cell type

Mesenchymal Stem Cells (MSCs)

Discovery

• Alexander Friedenstein discovered mesenchymal stem cells in mice (Mus musculus).
• From 1966 through 1987, Friedenstein provided evidence that stem cells from bone marrow can differentiate into mesenchymal tissues
• Since then, the cell potency of mesenchymal stem cells differentiation has been a cause of debate
• Are they truly multipotent or unipotent?

https://embryo.asu.edu/pages/mesenchyme

Defined

• Mesenchyme: loose cells embedded in the extracellular matrix
• a mesh of proteins and fluid that allows cells to migrate easily
• Directs development of morphological structures during the embryonic and fetal stages
• connective tissues, bones, cartilage, lymphatic and circulatory systems
• Carry over 480 growth factors and are attracted to target tissues of inflammation
• Primarily isolated from fat or bone marrow through a time-insensitive, invasive process
• Human fat (adipose tissue) has about 10x more stem cells than bone marrow

https://embryo.asu.edu/pages/mesenchyme

How They Work

• Derived from pluripotent stem cells, have already partially differentiated, and they continue specializing as they develop
• Must be activated appropriately – often mixed with human plasma
• Considered multipotent because their specialization potential is limited to one or more cell lines
• Current research suggests multipotent cells are able to go beyond the boundaries of producing one specific cell type but do so infrequently and only under narrow conditions

http://www.explorestemcells.co.uk/multipotentstemcells.html

• Modulate endogenous tissue and immune cells
• Actively interact with nearby cells
• Observed benefits of MSC therapy may result from the relinquishment of their molecular contents upon administration
• Therapeutic effects are short-lived
• “Recent studies have suggested that less than 1% of systemically administered MSCs persist for longer than a week following injection.”
• Limited in numbers – unlikely that true MSCs circulate peripherally (~0.01% of mononuclear bone marrow cells)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759519/

Clinical Indications

• Allogenic bone marrow transplants (from the same species to another of the same species) clinically used since the 1980s
• Autologous fat and bone marrow transplantations (from the same individual back to same individual) can be used to support target tissues that are not from the same cell type
• E.g., injecting fat MSCs into joints orthopedically to support the growth of new cartilage
• These MSC’s do not develop into new cartilage cells – they provide growth factors, reduction in inflammation, and immune modulation that may support joint health
• They are already on a development trajectory and their effects on unique target tissues are mostly paracrine (effecting nearby cells)
• Outside the US, MSCs can be cultured for several weeks to build up the cell counts
• The idea is that with more MSCs, more target tissues will benefit
• Challenge: they grow older the more times that they replicate so they are less effective
• MSCs are generally best used for transplantation into similar tissues from which they derive
• E.g., MSCs from fat are best transplanted into areas in need of fat replacement
• (breast augmentation, subcutaneous fat areas of the body – facial, lip, buttocks transplants)
• Most effective clinical use of MSCs:
• Same tissue transplantation (bone marrow to bone marrow, fat to fat)
• Joint conditions (if related to an autoimmune or systemic inflammation)
• Autoimmune disorders and systemic inflammatory conditions (see table on next slide)

Dangers and Side Effects

• Harvesting of bone marrow and fat MSCs is unpleasant for the patient
• There is a limited number of times one can extract and use fat MSCs
• Many patients have to repeat the procedure to gain significant benefit
• MSCs reduce inflammation for a time period of 6 months to 2 years but have limited regenerative benefits
• Are generally designed to affect one germ layer and tissues derived from that mesodermal layer – and under most conditions are unipotent (have the capacity to differentiate into only one cell type)
• Because of the immunomodulatory effects of these MSCs, they predispose the patients more infections or even cancer
• After MSC infusions were used to treat nine patients suffering from GvHD, three developed viral infections
• Immunosuppression by the MSCs had caused a reduction of immuno-surveillance to viruses

https://www.ncbi.nlm.nih.gov/pubmed/16604097

• MSCs, when administered in rats, can engraft in the renal tubules and mal-differentiate into adipocytes that hinder normal function of the kidney and lead to chronic kidney disease

https://www.ncbi.nlm.nih.gov/pubmed/17460140

Pluripotent Stem Cells

Discovery

• Henry Young et al. (2004) demonstrated connective tissue (including blood) contains reserve precursor cells
• Reserve precursor cells consist of: tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells
• Tissue-specific progenitor cells can be unipotent or multipotent
• Progenitor cells can only double 50–70 times while germ-layer lineage stem cells and pluripotent stem cells have a much greater lifespan
• Pluripotent stem cells were thought only to exist in embryonic stem cells until Dr. Young’s discovery of them in the peripheral blood in the late 20th century

Defined

• Precursor cells can be
• Tissue-specific progenitor cells
• Lineage-committed (ectodermal, mesodermal, and endodermal) germ-layer lineage stem cells
• Lineage-uncommitted pluripotent epiblastic-like stem cells
• What we are interested in today are the lineage uncommitted pluripotent stem cells  (some researchers call these cells, blastomere-like stem cells)

https://mail.google.com/mail/u/0/#search/henry+young+/13b421977b3e5c33?projector=1

How They Work

• Understanding lineage uncommitted pluripotent stem cells requires an understanding of the germ layers
• Adult pluripotent stem cells can be induced to form cells from the three primary germ-layer lineages (i.e., ectoderm, mesoderm, and endoderm).
• Results from neuronal, hematopoietic, diabetic, chondrogenic, osteogenic, myogenic, and cardiogenic studies demonstrate that adult pluripotent stem cells can be induced to undergo directed lineage induction.
• The activation of quiescent precursor cells is a potential component of tissue restoration.
• Quiescent stem cells also assist the tissue-committed progenitor cells in forming the missing tissues
• Originate in bone marrow and present in peripheral blood
• Contain a unique marker that can be used to select them for both diagnostic and therapeutic procedures
• In abundance in peripheral blood and in reproductive tissue secretions

Clinical Indications

• Lineage uncommitted pluripotent stem cells can be used to form any tissues in the endoderm, mesoderm, or ectoderm
• Treatment of a wide range of degenerative diseases in both humans and animals including, but not limited to:
• Diabetes, osteoarthritis, osteoporosis and Alzheimer’s disease, to name a few, as well as regenerative applications associated with aging
• TBI studies: When used in conjunction with hyperbaric oxygen therapy, intranasal and IV pluripotent stem cells (derived from blood plasma), after activation, have been shown, in case studies, to positively address post-concussive symptoms secondary to TBI: memory, sleep, mental fatigue, mental clarity, libido, motor function and balance.
• Could be shown useful in replacing bone marrow in post-cancer treatment

Making a stem cell decision

• Bone Marrow
  • Cost: $3,000 – $10,000
  • Recovery time: One Month
• Adipose (Fat)
  • Cost: $6,000 – $15,000
  • Recovery time: One Month
• Blood Based
  • Cost: $3,500 – $4,000
  • Recovery time: Less than a week

References

Cell Applications. https://www.cellapplications.com/stem-0

Cellular Differentiation. http://oerpub.github.io/epubjs-demo-book/content/m46036.xhtml

Kunter, U., Rong, S., Boor, P., Eitner, F., Müller-Newen, G., Djuric, Z., … & Milovanceva-Popovska, M. (2007). Mesenchymal stem cells prevent progressive experimental renal failure but maldifferentiate into glomerular adipocytes. Journal of the American Society of Nephrology, 18(6), 1754-1764.

MacCord, K. (2012). Mesenchyme. The Embryo Project Encyclopedia. Retrieved from https://embryo.asu.edu/pages/mesenchyme

Mesenchymal Stem Cell Reagents. Retrieved from http://www.sigmaaldrich.com/life-science/cell-biology/cell-biology-products.html?TablePage=22692887

Murnaghan, I. (2016). Multipotent stem cells. Explore Stem Cells. Retrieved from http://www.explorestemcells.co.uk/multipotentstemcells.html

Parekkadan B, Milwid JM. Mesenchymal Stem Cells as Therapeutics. Annual review of biomedical engineering. 2010;12:87-117. doi:10.1146/annurev-bioeng-070909-105309.

Stout, C. L., Ashley, D. W., Morgan, J. H., Long, G. F., Collins, J. A., Limnios, J. I., … & Young, H. E. (2007). Primitive stem cells residing in the skeletal muscle of adult pigs are mobilized into the peripheral blood after trauma. The American Surgeon, 73(11), 1106-1110.

Sundin, M., Örvell, C., Rasmusson, I., Sundberg, B., Ringden, O., & Le Blanc, K. (2006). Mesenchymal stem cells are susceptible to human herpesviruses, but viral DNA cannot be detected in the healthy seropositive individual. Bone marrow transplantation, 37(11), 1051-1059.

Tithon Biotech (n.d.). Minutevideo retrieved from http://mv.pac.io/post/55b2e2b41e1818650cc7b872

Tithon Human Sciences (2015). Peripheral blood derived pluripotent stem cell technology. Rhttps://aspenintegrativemedicine.com/wp-content/uploads/Tithon-Human-Sciences-Ortho-Case-Study.pdf

Young, H. E., Duplaa, C., Romero-Ramos, M., Chesselet, M. F., Vourc’h, P., Yost, M. J., … & Tamura-Ninomiya, S. (2004). Adult reserve stem cells and their potential for tissue engineering. Cell biochemistry and biophysics, 40(1), 1-80.

Young, H. E., & Black, A. C. (2004). Adult stem cells. The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology, 276(1), 75-102.