Ch 3 Stem cells and regenerative medicine

Question 1

4 classes of stem cells

stem cells are responsible for growth, maintenace and repair

Answer 1

embryonic ESC

Adult ASC

fetal/perinatal PSC

Induced pluripotent (IPSC) artificial

Question 2

potenency types

Answer 2

Totipotent; differentiate into cells from each of the germ layer lineages (zygote)

pluripotent; more than one germ layer lineage but not the extraembryonic (ESC and PSC)

multipotent; restricted to the germ layer lineage from which they originate (ASC)

Question 3

embryonic stem cells
What is? What problem?

Answer 3

harvested from the inner cell mass of a blastocyst, esulting in the destruction of the embryo

strong tendency to expand and differentiate in a dysregulated manner > resulting in the formation of teratomas

tumorigenic potential of undifferentiated embryonic stem cells has raised serious concerns

Question 4

adult stem cells
What is? List 3 types of

Answer 4

source of replacement cells that drives tissue maintenance and repair

purified from dissociated tissue and expanded in culture to isolate and attain

hematopoietic stem cells, mesenchymal stem cells, and neural stem cells

Question 5

Perinatal SC
Where from? 3

Answer 5

arvested from the umbilical cord, amniotic fluid, and fetal membranes

uperior expansion potential, increased plasticity, and may possess superior immune privilege

Question 6

induced pluripotent SC
What is? How generated?

Answer 6

directly reprogramming adult somatic (i.e., terminally differentiated) cells. Epigenetic reprogramming alters or “resets” the gene expression

First induced pluripotent stem cells were generated using a retroviral vector

risk of causing insertional mutagenesis or oncogenic transformation

Question 7

mesencymal SC

where located?

properties affected by wat variables?

what differntiate into? (3)

Answer 7

heterogeneous population of spindle-shaped, plastic adherent cells

reside in a perivascular location
> derived from either the bone marrow or adipose

cells within an individual colony of mesenchymal stem cells are morphologically diverse: small fast cells, large slow cells

capable of differentiating into cells such as osteoblasts, chondrocytes, and tenocytes

Question 8

Properties of MSC? (4)

Answer 8

1) adhere to tissue culture plastic and exhibit a spindle-shaped

(2) form colonies of cells from single parent cells when cultured in low-density “clonal” cultures (CFU)

(3) express a specific set of cell surface marker proteins (exclude from hematopoietic lineages)

(4) possess the ability to differentiate into osteoblast, adipocytes, and chondrocytes (i.e., tri-lineage differentiation) using defined in vitro differentiation assays

Question 9

msc mechanisams of action (4)

what anti-inflamm agents?

how immunosuppress?

Answer 9

terminally differentiating into various somatic cells “mesengenic process”

homing to sites of injury and contribute to tissue repair (migration and invasion) - paracrine effect.

Antiinflamm:
DAMP > macrophage (PRRs) > Il1a, TNF > MSC releases:
tumor necrosis factor-α (TNF-α)–stimulated gene/protein 6 (TSG-6), interleukin-1 receptor antagonist (IL-1ra), and prostaglandin E2 (PGE2)

Immunosuppression
(downregulate immune repsonse) via through direct suppression of T-cell proliferation as well as by guiding antigen presenting cells away from a proinflammatory phenotype

Question 10

Volk et al

canine MSC require what to differentiate to bone in vitro

Answer 10

bone morphogenetic protein-2 (BMP-2)

important differences between mesenchymal stem cells isolated from various species,

Question 11

MSC sources (5)

Autologus vs allogenic (donar)

Answer 11

bone marrow (dogs)
adipose tissue (dos)
synovium
muscle
teeth

btained from different donors and tissue sources exhibit substantial differences in regard to their properties and function, and the source of mesenchymal stem cells should be thoughtfully considered.

Question 12

ability of MsC

Answer 12

differentiate into a variety of connective tissues (bone and cartilage) > suggests that they have great potential for orthopedic applications

enhance the inherent repair of tissues through secretion of trophic factors

Question 13

how generate msc population

Answer 13

culture expanded
purification and in vitro expansion in culture to attain clinically relevant numbers of cells
pros: expand sufficient cell numbers in culture, the potential to cryopreserve large quantities of cells for readministration, the ability to accurately assess dosage and viability, and, most important, the ability to perform in vitro characterization assays prior to administration

unprocessed, minimally manipulated preparations of tissues that can be rapidly generated at the point of care
one marrow concentrate (BMC)141 or the stromal vascular fraction (SVF) of tissue digests
not possible to ascertain the absolute mesenchymal stem cell number, cell dose, or, in many cases, viability prior to clinical application

Question 14

hat are the options of MSC administration?

Answer 14

Intravascular infusion (cytotherapy) - less than 5%of cells successfully migrate to location of injury and survive

Direct implantation/injection (eg, seed onto a scaffold, intra-articular injection) ‘tissue engineering’

Question 15

main limitations/concerns regarding MSC therapy (8)

Answer 15

Gaps in knowledge

Inherent variability in product

Access to facilities and expertise

Increasing federal oversight (FDA has banned in humans in many states)

Unknown safety - stimulate growth of neoplasms?

Increased risk of infection?

Administration of IV bolus has risk of microvascular embolisation and ischaemia

Current lack of strong evidence

Question 16

Kadiyala et al…

Answer 16

First describes osteogenic and chondrogenic differentiation of bone marrow derived canine MSC

Question 17

2005, Volk et al…..

Answer 17

Evaluated osteogenic differentiation potential of marrow-derived MSC in 19 dogs - necessary to supply with BMP-2

Question 18

2008, Neupane et al…

Answer 18

completed most comprehensive characterisation of adipose-derived mesenchymal cells. Tend to proliferate more rapidly, preparation na dhandling effects proliferation rates

Question 19

2007 Black et al…

Answer 19

Evaluated effect of single intra-articular injection of stromal vascular fraction celss in 18 dogs with hip OA - significant improvement in lameness and function at 3m

Follow-up study - Single injection in 14 dogs with elbow OA - outcomes improved 30-40% comapred to baseline

Question 20

Nishida et al…

Answer 20

Weekly injections of bone marrow derived MSC into spinal cord lesion causing lack of deep nociception. No complications but no improvement

Question 21

Penha et al….

Answer 21

treated 4 dogs with longstanding neuro dysfunction from IVDD with intralesional bone marrow derived MSC. Reportedly improvements at 18m but not changes on MRI and were also having long-term physio

Question 22

Studies evaluation MSC treatment in cats…

Answer 22

Quimby et al - Safetly of systemically administered adipose derived stem cells to cats with CKD - adverse rxn from allogenic thawed cells at higher dose

Webb et al - allogenic MSC for Tx of chronic enteropathy. 5/7 significant improvement or resolution

Arzi et al - Cats with chronic gingivostomatitis Tx with 2 injections of autologout adipose derived MSC. 5/7 either complete remission or sunstantial improvement.

Question 23

Olsen 2019 VCOT

Answer 23

intravenously administered
allogeneic mesenchymal stem cells were well tolerated
by dogs with elbow osteoarthritis. While some subjective
outcome evaluations found significant improvements with
this treatment, a raft of different objective outcome measures
failed to demonstrate any significant improvement.

Question 24

Pavarotti 2020 VCOT

lack of a placebo control group

Answer 24

Intra-articular injection of autologous adipose tissue may
therefore represent a promising treatment for osteoarthritis
in dogs. The one-step procedure appeared safe, minimally
invasive and simple, with early and sustainable clinical
effects up to day 180.

Question 25

Pueckler 2022 VCOT

Answer 25

Answer 26

positive data suggesting both
in vitro and in vivo benefits of licensed MSCs, their
utilization for the treatment of tendon injury has
been greatly understudied. As described above,
2 murine studies28,75 have provided data that licensing
can enhance MSC therapy of an Achilles tendon
defect and stimulate tenocyte expression of
tendon-relevant genes.

Question 27

taguchi 2021 AJVR

Answer 27

Question 28

depompeo 2020 Vet surg

Answer 28

Additional studies are required to determine the relative
concentrations of viable AD-MSC obtained via traditionally
and laparoscopically harvested falciform tissue.
Furthermore, complete characterization of the cells and evaluation
of maintenance of multipotency and viability through
passage numbers

Question 29

Teunissen 2020 Vet J

several key questions about Achilles tendon injuries in humans and dogs become apparent and may account for the lack of MSC evidence in these species to date.41 Achilles tendon injuries in both species are commonly chronic and degenerative, so would they have the same inflammatory environment as an acute SDFT core lesion in the horse? If not, perhaps in vitro MSC licensing would be even more critical to improve treatment outcomes, as the MSCs would not otherwise be receiving the optimal endogenous signals from the injury environment

Answer 29

Canine MSCs (cMSCs) have been less extensively characterised
than hMSCs (de Bakker et al., 2013). Particularly, the chondrogenic
differentiation of cAT-MSCs is challenging; chondrogenesis in
earlier studies was either unsuccessful (Russell et al., 2016),
inferior to that of cBM-MSCs (Reich et al., 2012; Bearden et al.,
2017), or lacked robust evidence (Robey, 2017).

Question 30

he lack of control groups in publications that make efficacy claims is particularly problematic, as it was recently demonstrated that 39.7% and 79% of dogs in the placebo control arm of some veterinary clinical trials involving client-owned dogs experienced improved clinical signs (a phenomenon termed the placebo effect)

Answer 30

many of these reports are case series, involve small numbers of subjects, and are insufficiently powered to make conclusions regarding efficacy. In addition, many studies do not utilize an appropriate control group, fail to stratify patients properly between control and treatment arms, or utilize subjective outcome measures that are more likely to be affected by bias.

Question 31

Regenerative medicine is an interdisciplinary field of research and clinical therapy focused on repair, replacement, or regeneration of cells, tissues, and organs, with the goal of restoring structure and function to the affected tissue or organ.49 Regenerative medicine encompasses a wide variety of treatment strategies, including stem cell therapy, tissue engineering, gene therapy, and the use of autologous blood derivatives such as platelet-rich plasma or autologous conditioned sera.

Answer 31

clinical objectives of regenerative medicine include the reduction of inflammation and pain, improved healing of injured tissues, and ultimately return to function