Potency evaluation of tissueengineered and regenerative medicineproductsKelly Guthrie, Andrew Bruce, Namrata Sangha, Elias Rivera, and Joydeep Basu

Process Research and Translation, Tengion, Inc., 3929 Westpoint Blvd., Suite G, Winston-Salem, NC 27103, USA

Review

Glossary

Action-at-a-distance: mechanistic pathways operating between cells via, for

example, secreted paracrine factors, exosomes, and microRNAs that explain

how cell based therapies mediate regenerative outcomes independently of

site-specific engraftment and directed differentiation.

Active biological ingredient (ABI): the functional component of a medicinal

product that is responsible for mediating observed therapeutic outcomes.

Bioassay: a functional assay leveraging a living system (typically cell based,

although it may be animal-based) to quantifiably define the biologically

relevant activity of a therapeutic product candidate.

Critical quality attributes (CQAs): a set of key, quantifiable parameters used by

the US Food and Drug Administration to provide analytic definition of a

therapeutic product candidate.

Mechanism of action (MOA): the combination of factors, biological pathway, or

Methodologies for the rigorous and quantitative evalua-tion of biological activity or potency are an essentialaspect of the developmental pathway for all biologicproduct candidates. Such assays typically leverage keymechanistic pathways demonstrated to mediate ob-served therapeutic outcomes. Tissue engineered/regen-erative medicine (TE/RM) therapeutics include cell basedtherapies as well as engineered tissues and neo-organsfor which clarity regarding the mechanism or mecha-nisms of action may not be forthcoming. Here, we dis-cuss how strategies for the development of potencyassays for TE/RM product candidates may harness po-tential mechanisms of action or other therapeuticallyrelevant bioactivity along with cell number and viability.As the pipeline for TE/RM product candidates expandsthrough 2014 and beyond, the establishment of a definedframework for potency assays will facilitate successfultranslational outcomes.

Tissue engineered/regenerative medicine products arecomposed of cells and biomaterialsThe search for bioactive agents capable of modulatingdisease and catalyzing the body’s inherent ability to repairitself has evolved from the identification, isolation, andapplication of biologically derived or chemically synthe-sized small molecules and protein-based biologics to theconceptual recognition that the cell itself may be regardedas an active biological ingredient (ABI; see Glossary).Mechanistically operating in part through action-at-a-dis-tance paracrine signaling pathways, the cell in its capacityas a therapeutic agent may serve to recruit and mobilizenative (i.e., host derived) stem and progenitor cell popula-tions, or it may release growth factors and exosomes thatpromote angiogenesis and neurogenesis, modulate inflam-matory, fibrotic, and apoptotic cascades, and generallyfunction to interfere with the onset of pathology whilepromoting self-repair and regeneration. Furthermore,the cell as medicinal agent may itself directly contributetowards the regeneration of native tissues and organs byniche-specific directed differentiation towards defined de-velopmental lineages as regulated by contextual signaling

0167-7799/$ – see front matter

� 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tib-

tech.2013.05.007

Corresponding author: Basu, J. (joydeep.basu@tengion.com).Keywords: potency; cell therapy; tissue engineering; regenerative medicine.

cues derived from the surrounding tissue or organ paren-chyma [1].

Any tissue engineered/regenerative medicine (TE/RM)product or product candidate incorporating a cellular ABImay be defined as a bi-component ‘construct’ composed of:(i) Cellular ABI (primary, lineage committed cell, pro-

genitor, or stem cell population).(ii) Biomaterial or scaffold. In its broadest sense, the term

biomaterial may be used to refer to a basal diluentsuch as buffered saline.

Acellular biomaterials, such as small intestine submu-cosa (SIS), have also been developed into successful TE/RMproducts [2].

The role of biomaterials within TE/RM products hasevolved from merely providing a passive structural frame-work within the body to facilitate repair or regeneration (asfor example with the use of gold or porcelain to repairdental cavities) towards the development of more bioactivebiomaterials through integration with defined stem orcommitted cell populations. Such bioactive biomaterialscan form regenerative scaffolds that are capable of facili-tating the deposition of extracellular matrix (ECM) bytherapeutically bioactive cell populations as well as medi-ating the formation of a regenerative milieu to catalyze theinduction of neo-tissues or neo-organs. In addition, thebiomaterial scaffold may serve to recapitulate aspects ofthe complex, 3D microarchitecture of the targeted organ.

network of biological pathways by which a medicinally bioactive material

mediates observed therapeutic outcomes.

Neo-organ: a regenerated organ made by tissue-engineering techniques.

Potency: the specific ability or capacity of a product, as indicated by appropriate

laboratory tests or by adequately controlled clinical data obtained through the

administration of the product in the manner intended, to effect a given result.

Trends in Biotechnology, September 2013, Vol. 31, No. 9 505

Review Trends in Biotechnology September 2013, Vol. 31, No. 9

Alternatively, the biomaterial may actively interact withthe cell in a manner that mimics signaling communicationsbetween cell and ECM during early embryonic develop-ment and organogenesis. Ultimately, the biomaterial scaf-fold will degrade completely and coordinately with thedeposition of ECM and the development of laminar orradial multicellular organization within the regeneratingneo-organ.

Critical quality attributes of TE/RM product candidatesCurrent regulatory paradigms for the characterization ofmedicinal products are tailored principally towards smallmolecule chemical entities and biopharmaceuticals, suchas natural or recombinant-sourced proteins. As the empha-sis in therapeutic product research evolves away from theapplication of the cell as a mere manufacturing platformtowards a realization of the cell itself as the manufacturedproduct, it is imperative that the emerging class of novelTE/RM therapeutics is accommodated within suitably flex-ible frameworks for process and manufacturing, qualitycontrol, supply chain management, toxicological and path-ological evaluation, therapeutic bioactivity evaluation,preclinical animal studies, and clinical trials. Alternative-ly, existing policies and regulatory infrastructure willrequire reformulation to remain relevant, permitting theappropriate governmental agencies to effectively deliveron their mission to facilitate approval of safe and effectivenovel therapeutic products without needlessly burden-some oversight of the nascent TE/RM industry.

To this end, the US Food and Drug Administration(FDA) guidelines (www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm) identify four principal critical quality attri-butes (CQAs) for the analytic definition of cell based ther-apeutics: identity, purity, safety, and potency. Theevaluation of identity is required to establish the presenceof bioactive cellular components. Measures of purity con-firm the absence of undesirable elements of any nature,including cellular contaminants and process chemicals.Safety evaluation should demonstrate the absence ofbio-burden (such as contaminating bacteria, fungi, proto-zoa, and viruses) and lack of tumorigenic potential. Finally,potency represents an index of the product’s therapeuti-cally relevant biological activity [3,4].

As progressively more TE/RM product candidates areentering into clinical trials [5,6], there is a considerableunmet need within the TE/RM industry for agreement onthe definition of pertinent common regulatory frameworks.Movements by industry working groups towards the iden-tification of standardized analytical methodologies forcharacterizing the potency of TE/RM products have typi-cally focused on the identification and optimization ofanalytic techniques relevant to stem cell based productcandidates, such as mesenchymal stem cells (MSCs) andhematopoietic stem cells [7]. These efforts notwithstand-ing, clarity regarding the implementation of CQA criteriaremains forthcoming for tissue-engineered products suchas the Neo-Urinary Conduit (NUC; Tengion), Neo-BladderReplacement (NBR; Tengion), and Neo-Kidney Augment(NKA; Tengion), as well as neo-vessels and other engi-neered and regenerated neo-organs and tissues [5]. As the

506

NUC and NKA represent the vanguard for a new genera-tion of TE/RM product candidates entering clinical trials,we will leverage these as illustrative examples for regula-tory agencies and industry working groups in the TE/RMspace to provide leadership and direction as to how potencyassays for these novel products should be developed andimplemented.

PotencyPotency is ‘the specific ability or capacity of the product, asindicated by appropriate laboratory tests or by adequatelycontrolled clinical data obtained through the administra-tion of the product in the manner intended, to effect a givenresult’ (21 Code of Federal Regulations, Part 600.3(s),www.fda.gov/BiologicsBloodVaccines/GuidanceComplian-ceRegulatoryInformation/Guidances/default.htm). Bytheir nature, TE/RM therapeutics pose inherently uniquechallenges for potency assay development and implemen-tation. These challenges include: the heterogeneous natureof both the sourcing material (typically human tissues ororgans from allogeneic or autologous donors) and the prod-uct itself; the novelty of the product and the concomitantlack of knowledge regarding its possible mechanism ofaction/mechanisms of action (MOA/MOAs) and in vivobiodistribution; the absence of appropriate establishedreference standards and controls; and constraints regard-ing the availability and stability of product for potencytesting [4]. Ideally, potency testing is applied to the productas administered with all its components. However, thismay not always be feasible for the reasons just outlined.Custom-fabricated, tissue-engineered products — such asthe NUC or NBR — that are produced in a closed bioreactorfor individual patients from autologous-sourced tissue maybe especially problematic.

The potency of any biological product is typically mea-sured through application of a quantitative biological as-say (bioassay) that measures the activity of the product asdefined by its specific ability to effect a particular outcome.Bioassays are characterized by the leveraging of livingexperimental systems (in vivo animal models, in vitrotissue, organ cultures, or cell cultures) to establish theactivity of a therapeutic product. By contrast, analyticalassays evaluate the properties of a product candidateoutside a living system, as with ELISA, fluorescence-acti-vated cell sorting (FACS), and PCR for example. A combi-nation of bioassay and analytical assays may be required toadequately characterize the potency of a cell based thera-peutic [3].

Guidance to industry on potency evaluation of TE/RM

products

The FDA recommends (www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm) that the ideal potency assayshould have the characteristics listed in Table 1. However,for most cases, it is unlikely that any one potency assay byitself will present this complete profile. Therefore, the FDArecommends the assembly of a potency bioassay matrixcomposed of multiple functional bioassays that, takentogether, address most or all of the points in Table 1.The combination of assays within the assay matrix serves

Table 1. Key features of a potency assay, as recommended bythe US FDA

(i) Provides a demonstration of functional bioactivity relevant to a

known or postulated mechanism of action

(ii) Is appropriate for product lot release

(iii) Is specific to the product

(iv) Is quantifiable

(v) Can demonstrate lot-to-lot consistency

(vi) Has been validated, i.e., meets pre-defined acceptance/rejection

criteria

(vii) Is comparable to a reference standard

Review Trends in Biotechnology September 2013, Vol. 31, No. 9

to address all of the FDA’s requirements. Within thematrix, a broad variety of biological and analytical tech-niques may be leveraged.

An iterative, progressive approach towards the devel-opment and implementation of potency assays is recom-mended, with the understanding that assay methodologywill be dynamic and will evolve with the phase of productdevelopment as a solid database of product characteristicsand potential MOAs becomes established over time. Thepredictive potential of potency bioassays with regard toclinical efficacy and outcomes is contingent on such adatabase. To this end, multiple bioassays leveraging thebroadest possible range of product characteristics and(potential) MOAs should be explored in parallel from theearliest stages of product development. Data on the tran-scriptomic (including microRNA), proteomic, secretomic,immunological, biophysical, and other phenotypic charac-teristics of the product should be compiled on candidateproduct lots within a single database. Cross-referencingthese data with in vivo studies on MOAs and/or previouslypublished studies on MOAs of similar cell based therapeu-tics will typically identify potential avenues for potencyassay development. The FDA will ‘‘allow for considerableflexibility in determining the appropriate measure of po-tency for each product’’ [8].

Relationship of cell count/viability to potency

Cell count/viability is an inadequate marker of productpotency in the absence of supporting bioassay data specifi-cally interrogating potential MOAs. Cells may be ‘viable’ asdetermined by metabolic assays but show substantiallyreduced biological activity. For example, the potency ofviable MSC cultures as measured by tri-lineage differenti-ation typically decreases with passaging or with increasingage of the donor [9]. Nevertheless, measurement of cellcount/viability is needed to ensure potency is expressed inthe form: functional unit per viable cell, and cell count/viability is therefore typically included as a componentwithin the potency assay matrix (Boxes 1–3).

A potency assay should ideally leverage a known aspect

of product MOA, as identified through in vivo studies

The multi-modal MOAs by which TE/RM products arepresumed to mediate therapeutic bioactivity complicateefforts to establish relevance for potency assay develop-ment. Functional paradigms are subject to constant fluxand re-evaluation. For example, directed differentiationand site-specific engraftment of MSC and MSC-like cellpopulations is no longer thought to represent the principal

pathway for their clinically relevant functional bioactivity[10]. Current ‘touch-and-go’ models for the MOAs of MSCsemphasize paracrine bioactivity and direct cell–cell contactto explain the observed action-at-a-distance therapeuticeffect of MSCs [11]. Consistent with this hypothesis, rodentmodels of inflammatory organ failure show increases insurvival and modulation of inflammation on administra-tion of MSC-derived conditioned media (MSC-CM) and/orMSC cellular lysates with concomitant increases in seruminterleukin-10 (IL-10). This effect may be mimicked bytreatment of peripheral blood mononuclear cells (PBMCs)in vitro with MSC-CM, forming the basis of a specificpotency assay for MSC for this particular therapeuticindication. Absolute amounts of secreted IL-10 derivedfrom treatment of PBMC in vitro with MSC-CM may thenbe quantified by ELISA [12].

Similarly, secretion of hepatocyte growth factor (HGF)has been directly linked to observed MSC-mediated func-tional rescue in animal models of multiple sclerosis, sug-gesting that HGF levels in cultures of MSC may beleveraged as a surrogate indicator of product potency [13].Additionally, secretion of vascular endothelial growth factor(VEGF) and kidney injury molecule 1 (KIM1) is currentlyleveraged as a potency bioassay for the bioactive cellularcomponent of the NKA [14]. Finally, secretion of VEGF hasbeen reported as a potency metric for ex vivo-produced oralmucosal equivalent (EVPOME), a tissue-engineered, cellbased product prototype for intra-oral grafts [15].

Additional mechanisms of cell–cell communication in-volving action-at-a-distance signaling interactions be-tween MSCs and non-stem cell populations identifyfurther avenues for potency assay development. In partic-ular, considerable focus is being placed on the role ofexosomes and microvesicles in mediating aspects of cell–cell signaling and interaction. Microvesicles are circularmembrane fragments that are found in numerous biologi-cal fluids, and they might mediate the horizontal cell–celltransfer of functional proteins and/or genetic informationin the form of microRNAs that are potentially capable ofreprogramming injured cells towards dedifferentiation,proliferation, and subsequent expression of trophic factorsthat facilitate the targeted recreation of a regenerativemicroenvironment [16]. The identification of therapeuti-cally relevant signaling pathways modulated by secretedcytokines or microvesicles may be accelerated by leverag-ing array-based cellular reporter platforms that directlymonitor activity of the principal transcription factors mo-bilized by these signaling elements (see ‘Cell-Based Assaysfor Pathway Analysis’ on the SABiosciences website(www.sabiosciences.com/cellassay.php)). In turn, thesehighlighted pathways may form the foundation for cellbased functional bioassays. Examples illustrating howspecific or presumed MOAs may be leveraged for bioassaydevelopment in TE/RM products and product candidatesare presented in Table 2.

Potency surrogates

Bioassays by definition entail the application of livingsystems, typically cell cultures, to provide a functionalreadout of product potency. It is universally understoodthat the use of living systems is associated with unique

507

Box 1. Apligraf

Apligraf is a tissue-engineered, bi-laminar, skin-like construct currently

on the market for the treatment of recalcitrant ulcers. Compositionally,

Apligraf consists of allogeneic-sourced human epidermal keratino-

cytes organized into a skin-like, cornified upper layer and a lower layer

composed of allogeneic-sourced human dermal fibroblasts sus-

pended in a bovine collagen matrix (Figure I). Mechanistically, Apligraf

is understood to mediate wound healing by providing a physical

barrier to the wound surface while recreating a regenerative milieu

through secretion of regenerative growth factors such as platelet-

derived growth factor, fibroblast growth factor, and epidermal growth

factor. The potency of Apligraf is assessed through quantitative

morphological evaluation of sample product from a given product lot.

The potency assay for Apligraf consists of a battery of histological

parameters that, taken together, are diagnostic of an appropriately

manufactured, viable, and functional product:

(i) Epidermal coverage: the surface area of product covered by

epidermis.

(ii) Epidermal development: the presence of a basal layer of

cuboidal/columnar keratinocytes, at least five stratified supraba-

sal layers, and at least one cornified squamous cell layer on the

apical surface.

(iii) Basal keratinocyte cell viability: the surface area of epidermis with

viable basal keratinocytes, as defined by the presence of

basophilic cytoplasm without severe vacuolization or necrosis.

(iv) Suprabasal keratinocyte cell viability: the proportion of supraba-

sal keratinocytes containing basophilic cytoplasm without va-

cuolization, necrosis, or pyknosis.

(v) Dermal thickness: the thickness of dermal matrix across the

product, as evaluated by random sampling.

(vi) Fibroblast density: the mean fibroblast density (viable, non-

pyknotic nuclei) in random sampling of dermal matrix.

(vii) Matrix aspect: the percentage of dermal matrix collagen that is

uniformly stained without large holes or inclusions.

Each of these morphological parameters serves as a surrogate

marker of functional bioactivity established both in vitro and in vivo.

Key product-related functional outcomes include: physical integrity

and impermeability to water; cell metabolic activity {as measured by

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]

assay}; secretion of the pro-angiogenic growth factor vascular

endothelial growth factor; cellular viability; and growth rate during

manufacture and morphological assessment of grafts implanted in

vivo within rodent models. More details regarding key studies

demonstrating linkage between histological indices and functional

bioactivity are presented in the briefing documents of the Cellular,

Tissue and Gene Therapies Advisory Committee Meeting, November

17, 2011 (http://www.fda.gov/AdvisoryCommittees/Committees

MeetingMaterials/BloodVaccinesandOtherBiologics/cellular

TissueandGeneTherapiesAdvisoryCommittee/ucm279851.htm).

(A) Human skin (B) Apligraf

Stratum corneum

Dermal matrix withfibroblasts

Basal layer

TRENDS in Biotechnology

Figure I. Comparison of (A) human skin with (B) Apligraf, showing reconstitution of laminar organization. Evaluation of Apligraf histoarchitecture is the basis of the

potency assay for Apligraf.

Review Trends in Biotechnology September 2013, Vol. 31, No. 9

technical challenges that may be bypassed through theapplication of an analytic assay or assay matrix as asurrogate marker of biological activity. This requires theestablishment of a clear and statistically sound correlationbetween biological activity and the nonbiologic analyticassay. Ultimately, the analytic assay may be leveraged asan expedient alternative to the original bioassay for mea-surement of potency. For example, levels of the cytokinesVEGF, CXC chemokine ligand 5 (CXCL5), and IL-8 fromMSC-CM have been applied as surrogate potency markersthat are reflective of the pro-angiogenic bioactivity of MSCcell populations as observed in an in vitro endothelial cellbased angiogenesis assay [17]. Through correlation of thelevels of cytokine required to induce tube formation in vitrowith the levels of cytokine found in spent media frommanufacturing production runs, the detection of these

508

factors was identified as a surrogate potency assay withdefined pass/fail criteria. De novo angiogenesis is hypothe-sized to be one possible MOA for MSC bioactivity in animalmodels of myocardial infarction [18]. Finally, quantitativehistological evaluation of multiple morphologic param-eters has been leveraged as a surrogate potency indexfor Apligraf (Organogenesis), a tissue-engineered skin sub-stitute, as detailed in Box 1.

Potency assays for tissue engineered neo-organs and

neo-tissues

Neo-organs and neo-tissues composed of bioactive cellpopulations complexed with biomaterial scaffolds repre-sent an entirely novel class of TE/RM products. Suchproducts have limited case histories for the provisionof guidance to regulators and industry regarding the

Review Trends in Biotechnology September 2013, Vol. 31, No. 9

development and implementation of potency assays (seethe survey of products and product candidates in the TE/RM space in Table 2). Examples of products in the marketinclude Apligraf and Dermagraft (Shire Regenerative Med-icine) (Box 1) [19]. The NBR, NBA, and NUC represent thefirst tissue-engineered human neo-organ product candi-dates to reach Phase I/II clinical trials [5] and as suchfunction as illustrative examples for the field (Box 2). Thesebladder-based neo-organs are generated from autologous-sourced smooth muscle cells seeded onto synthetic, biode-gradable polymers [20]. This binary construct is maturedex vivo within a closed bioreactor to facilitate cell prolifer-ation and deposition of ECM upon the scaffold. At time ofsurgery, the matured construct is wrapped within omen-tum and connected to the patient’s uro-genital infrastruc-ture as appropriate (Box 2) [21]. The presence of omentumaround the implanted product is critical to successfulregenerative outcomes and neo-organ induction [22].

The spatiotemporal sequencing of histogenesis duringhollow neo-organ regeneration has been characterized inporcine cystectomy models showing regeneration of neo-urinary tissues from implanted NUC [23]. As seen inFigure I in Box 2, at 6 days after NUC implantation,migration of mesenchymal and endothelial cell populationsfrom omentum through the NUC construct towards theluminal surface is clearly visible. Developing neovascula-ture is also evident. These cell populations are likely to bederived from the omentum [24]. Similarly, the urothelialcells populating the luminal surface of the construct mustbe sourced from the ureters (see Figure I in Box 2).

These observations implicate the migration of host-de-rived cell populations as a principal MOA underlyingregenerative outcomes in neo-organ formation, therebyidentifying cell migration as a specific functional bioassay

Box 2. Neo-urinary conduit and other hollow, tubular neo-organ

Regeneration of the trilaminar histoarchitecture of the Neo-Urinary

Conduit (NUC) is believed to be mediated by two principal mechanistic

pathways: paracrine signaling from the smooth muscle cell population

used to seed the scaffold and migration of host-derived progenitor

and committed cell populations. These putative mechanisms of action

form the basis of defined, quantifiable bioassays to evaluate NUC

product potency as outlined below.

Mechanism of action: paracrine signaling through secreted factors

such as VEGF and MCP1

Recruitment of host-derived cell populations, including smooth

muscle cells, endothelial cells, monocytes, and resident stem and

progenitor cells, during regenerative remodeling of implanted con-

structs is understood to be mediated in part by the secretion of

paracrine factors, including vascular endothelial growth factor (VEGF)

and monocyte chemotactic protein 1 (MCP1) [38].

Assay

A surrogate index of potency for the NUC can be obtained by

evaluating the presence of VEGF and MCP1 in spent media from NUC

bioreactors post-maturation (as quantified by ELISA) along with

measuring cell viability.

Mechanism of action: mobilization and migration of resident cell

populations

Tubular organ regeneration involves a specific temporal sequence of

cellular infiltration, vasculogenesis, and neurogenesis with concomi-

tant differentiation of mucosal, stromal, and parenchymal laminar

tissue architectures. By definition, migration of host-derived cell

that may be leveraged to evaluate the potency of the NUCand related hollow neo-organ constructs, including thesmall intestine and esophagus (Box 2) [25–27]. Migrationhas also been applied as a potency assay for bone-marrow-sourced MSCs, which are under development as a cellulartherapy for cardiac infarction [28]. The secretion of growthfactors, such as VEGF and monocyte chemotactic protein 1(MCP1), that mobilize host-derived cell populations formigration towards the regenerating neo-organ may beleveraged as a surrogate potency indicator. For tissue-engineered constructs that are acellular, the biomaterialitself is the bioactive ingredient. Examples of such con-structs include decellularized vascular grafts made byseeding synthetic, biodegradable tubular scaffolds withsmooth muscle cells that are subsequently removed bydetergent-mediated decellularization [29]. For such pro-ducts, the migration of host-derived cell populations ontothe construct is by definition the principal MOA and musttherefore serve as the basis for potency-related bioassays.

The NKA, a primary renal cell–hydrogel compositetherapeutic for the amelioration of end-stage renal disease,is hypothesized to function in part by site-specific engraft-ment and regeneration of new tubular structures withindiseased kidney [30]. Product potency may therefore beevaluated by in vitro bioassays that measure tubulogenicpotential: that is, the ability of product constituents tospontaneously assemble into tubular or spheroidal struc-tures upon culture in 3D hydrogels or chicken eggs [31,32].The number of tubular or spheroidal structures may benormalized relative to parameters such as the number ofcells, the volume of the hydrogel, or the number of micro-scope fields scored. Additionally, as with the NUC, theNKA is thought to function by stimulating the migration ofendogenous populations of stem and progenitor cells and

products

populations, such as neural cells, endothelial cells, and urothelial

cells, is a principal mechanism of action for NUC and NUC-like neo-

organ regeneration, as such cell populations are extensively distrib-

uted throughout the final, fully regenerated tissue despite not being

present in the implanted construct. Wrapping of construct in omentum

is critical for successful regenerative outcomes [5]. Omentum is well

established as a source of mesenchymal and other stem cell

populations with regenerative potential [24]. As shown in Figure I,

the preliminary stage in regeneration of neo-urinary-like tissue from

implanted NUC is the migration of vasculature and mesenchymal cell

populations from surrounding omentum through the NUC biomaterial

towards the luminal surface. Efficient cellular migration into the NUC

construct is contingent on the presence of smooth muscle cells (the

bioactive ingredient) within the construct. This functional outcome

forms the basis of a migratory assay for construct potency.

Assay

NUC constructs are custom fabricated from autologous-sourced

smooth muscle cells and biodegradable, synthetic polymers [5].

Because constructs are matured in a closed system and shipped

directly to the implant site, they cannot be sampled for potency

evaluation. Therefore, for each construct, a mini-NUC cassette

composed of biomaterial from the same lot as the actual product is

seeded with the ABI, the smooth muscle cells sourced from the patient

to be implanted [20]. As shown in Figure I, the migration of labeled

mesenchymal stem cells, endothelial cells, or other cell populations

from a hydrogel or other biomaterial source external to the mini-NUC

cassette (an in vitro surrogate for the omentum) onto the mini-NUC

cassette over a defined period of time may be quantified.

509

(A) (B)

(C)

L

TRENDS in Biotechnology

Figure I. Central panel: wrapping the NUC construct in omentum provides it with a source of vascularization. (A) Cartoon representation of observed infiltration and

migration of vascular structures and mesenchymal cell populations from the surrounding omental wrap into the NUC construct. The arrow represents the direction of

migration. (B) Histology (hematoxylin and eosin staining) of actual porcine NUC at 6 days post-implantation. Note the accumulation of remnant scaffold material (white

under polarizing light). The luminal surface (L) is towards the top of the panel. Note the cellular infiltration with intense fibrovascular response in the outer one-third of the

wall. The arrow represents the direction of migration. (C) Cartoon representation of the migration of urothelial cells from ureters into NUC lumen. As outlined above, the cell

migration of ectopic cell populations is the basis of the potency assay for NUC constructs.

Review Trends in Biotechnology September 2013, Vol. 31, No. 9

by facilitating de novo angiogenesis and amelioration offibrosis and apoptosis within the diseased kidney (Box 3)[33,14].

To this end, the development of tubulointerstitial fibro-sis during the progression of chronic kidney disease (CKD)is associated with transforming growth factor-b (TGFb)-mediated epithelial–mesenchymal transition (EMT) oftubular epithelial cells [34]. Attenuation of TGFb-relatedpathways has been observed in vivo in a rodent model ofprogressive CKD in which survival was extended andrenal function improved by treatment with NKA proto-types, establishing another potential in vivo mechanisticbioactivity for the NKA [35]. An in vitro model systemrecapitulating observed disease and regenerative out-comes may be assembled using the human proximal tubu-lar cell line HK2, which is well established as a cellularplatform to test the stimulatory or inhibitory effects ofsmall molecules or proteins on TGFb-induced EMT [36].Modulation of TGFb-induced EMT in HK2 cells hasbeen leveraged as a quantifiable potency assay for NKAcandidates [14].

Finally, as discussed previously, microRNAs have beendemonstrated to mediate cell–cell communication through

510

incorporation within microvesicles or exosomes and rep-resent one possible MOA to explain the action-at-a-dis-tance feature of many cellular therapies, including theNKA [16]. The nuclear factor-kB (NF-kB) signal trans-duction pathway is known to be progressively activatedwithin rodent 5/6 nephrectomy models of CKD [30].Intrarenal delivery of the NKA has been demonstratedto decrease NF-kB nuclear localization within the rodent5/6 nephrectomy model of CKD, providing a clear mecha-nistic pathway for product functionality [30]. An in vitrobioassay recapitulating this in vivo MOA has been devel-oped: conditioned media from the NKA attenuates tumornecrosis factor-a-induced NF-kB activation within HK2cells (a proximal tubule-derived cell line). Analysis of themicroRNA content of exosomes isolated from NKA-condi-tioned media by ultracentrifugation using PCR-basedarrays of known sequences identified several microRNAspecies that are potential repressors of NF-kB signaltransduction, including microRNA-21 (mir-21). There-fore, mir-21 represents a putative potency surrogatebiomarker for product-derived bioactivity associatedwith modulation of the NF-kB signaling pathway (seeIlagan et al. ‘Secreted factors from bioactive kidney cells

Box 3. Neo-Kidney Augment

The regeneration of kidney structure and function following implanta-

tion of the Neo-Kidney Augment (NKA) into diseased kidney is

hypothesized to leverage multiple mechanisms of action (MOAs),

including de novo tubulogenesis, paracrine bioactivity, modulation of

host epithelial–mesenchymal transition (EMT), mobilization and migra-

tion of host progenitor cell populations, and de novo angiogenesis.

Bioassays based on these mechanisms of action, as outlined below,

may be organized into a potency matrix to evaluate NKA bioactivity.

� MOA: regeneration of tubular function in recipient diseased kidney

by formation of new kidney tubules. A key functional characteristic

of the NKA-derived cell population is self-organization into 3D

cellular aggregates, such as spheroids, organoids, tubules, and

tubular networks, within 24 hours after culture in 3D hydrogels

composed of collagen I and/or collagen IV. These self-organized

structures present robust expression of key functional markers that

are diagnostic of tubular epithelial cells. The formation of tubules

and organoids can be scored and quantified (Figure I). Assay: de

novo self-organization of the product-derived cellular component

into tubules and organoids on culture in 3D hydrogels.

� MOA: paracrine bioactivity, including secretion of the regenerative

growth factors vascular endothelial growth factor (VEGF) and kidney

injury molecule 1 (KIM1), and microRNAs such as microRNA-21

(mir-21). Assay: qRT-PCR or fluorescence-activated cell sorting

(FACS) quantification of mir-21 expression from product candidate.

mir-21 expression can also be quantified in real time in living

cells by application of Smartflare probes (Millipore, www.millipore.

com/publications.nsf/a73664f9f981af8c852569b9005b4eee/

bac85571e429339b85257a98005694e8/$FILE/TN4254EN00_EM.pdf).

� MOA: mobilization and migration of host-derived progenitor cells

along the stromal cell derived factor 1 (SDF1)–CXC chemokine

receptor 4 (CXCR4) axis. The SDF1–CXCR4 axis is a well-established

pathway for mobilization and migration of native progenitor cell

populations [39]. Migration of fluorescently labeled product-derived

cell populations within a defined time window through a hydrogel

barrier in response to recombinant SDF1 is being developed as an

index of NKA potency (Figure I). Assay: migration of labeled

product-derived cell sample within a defined time window through

a hydrogel barrier in response to SDF1.

� MOA: modulation of fibrosis by interference with EMT events in

diseased host kidney. Assay: attenuation of transforming growth factor-

b-induced EMT in HK2 cells, monitored by qRT-PCR analysis of E-

cadherin (an epithelial marker) and calponin (a mesenchymal marker).

� MOA: de novo angiogenesis. Assay: self-organization of human

umbilical vein endothelial cells (HUVECs) into 2D tubular structures

in response to product-derived conditioned media. Product may

function in part by promoting the de novo assembly of vasculature

through secretion of pro-angiogenic factors (VEGF). Assembly of

tubular networks from HUVECs in 2D culture in the presence of

NKA-derived conditioned media is used as a cell based readout for

functional VEGF secreted by NKA (Figure I).

(A) (B)

(C)

(D)

TRENDS in Biotechnology

100µm

100µm

100µm

Figure I. MOA of the NKA. (A) Injection of cell–hydrogel composite into renal parenchyma of diseased kidney mediates multiple regenerative outcomes, including, from top to

bottom, de novo nephrogenesis, mobilization and migration of host-derived stem and progenitor cell populations, and paracrine-mediated signaling. (B) De novo tubulogenesis of

NKA renal cell populations in 3D collagen gel matrix. Self-organized tubules express diagnostic functional markers, including aquaporin 1 (green) and cytokeratins (red). Tubule

formation may be easily quantified/unit cell. (C) Media derived from NKA cell populations induces the self-assembly of HUVECs into 2D tubular networks, providing a quantifiable cell

based assay for the presence of pro-angiogenic paracrine factors such as VEGF. (D) Migration of NKA cell populations in response to a defined source of SDF1. Response to SDF1 is

associated with upregulation of CXCR4, the receptor for SDF1, shown in green. Both migration and CXCR4+ cell populations may be quantified as a cell based assay. Tubule

formation may be easily quantified and expressed as number of tubules formed per unit cell population used in the assay. For example, if 100 cells were used to seed the assay and 20

tubules were scored, the assay outcome may be expressed as 20/100 or 0.2 tubules/unit cell. Each of these MOAs may be leveraged as the basis of a potency assay as described.

Review Trends in Biotechnology September 2013, Vol. 31, No. 9

511

Table 2. Development of potency assays for TE/RM productsa

Product Product category Company Clinical or market status Mechanism of action Potency bioassay Refs

Neo-Urinary

Conduit

Tissue-engineered

neo-organ

Tengion Phase I, cystectomy

secondary to bladder

cancer/injury

(i) Paracrine secretion

of MCP1

(ii) Migration of host

derived cell populations

(i) ELISA for secretion

of MCP1

(ii) Cell migration in vitro

[25]

Neo-Kidney

Augment

Cell based therapy Tengion Phase I, chronic

kidney disease

(i) De novo tubulogenesis

(ii) Paracrine secretion of

VEGF and mir-21

(iii) Mobilization and

migration of host

cell populations in

response to SDF1

(i) In vitro tube formation

assay

(ii) HUVEC angiogenesis

assay; RT-PCR analysis

of mir-21

(iii) In vitro cell migration

and fluorescence

analysis of CXCR4

upregulation

[31]

Apligraf Tissue-engineered

skin graft

Organogenesis Market (i) Creation of physical

barrier

(ii) Paracrine bioactivity

Quantitative histological

analysis of product

structure

b

Dermagraft Tissue-engineered

skin graft

Shire

Regenerative

Medicine

Market Paracrine bioactivity ELISA for secretion of

regenerative factors

such as VEGF

[19]

Multistem Cell based therapy:

MSC-like cells

Athersys Phase I, cardiac

ischemia

Paracrine bioactivity ELISA for secretion of

VEGF, IL-5, and CXCL5

[17]

Prochymal Cell based therapy:

MSC

Osiris Phase III, graft versus

host disease

Paracrine bioactivity ELISA for secretion of

TNFR1

[11]

GRN001 Cell based therapy:

hESC-derived

oligodendrocyte

progenitors

Geron Phase I, neural repair,

spinal cord injury

Paracrine bioactivity ELISA for secretion of

neurotrophic factors

[21]

Amorcyte,

AMR001

Cell based therapy:

CD34+CXCR4+ cells

Neostem Phase I, myocardial

infarction

Mobilization and migration

of product to damaged

tissue along SDF1 gradient

In vitro migration of

CD34+CXCR4+ cells

in SDF1 gradient

[39]

Chondrocelect Cell based therapy:

autologous

chondrocytes

TiGenix Market In vivo chondrogenesis

and cartilage formation

PCR-based evaluation

of marker expression

[7]

Provenge Cell based therapy:

primed, expanded

T cells

Dendreon Market Activated T cells trigger

FAS-mediated apoptosis

of leukemic B cells

FACS analysis of CD54 [40]

MA09-RPE Cell based therapy:

ESC-derived retinal

pigment epithelial

cells

Advanced

Cell Therapy

Phase I, macular

degeneration

Phagocytosis and clearing

of cellular waste

Phagocytosis of

fluorogenic bioparticles,

FACS analysis

[41]

aAbbreviations: CXCL5, CXC chemokine ligand 5; CXCR4, CXC chemokine receptor 4; ESC, embryonic stem cell; FACS, fluorescence-activated cell sorting; hESC, human

ESC; HUVEC, human umbilical vein endothelial cell; IL-5, interleukin-5; MCP1, monocyte chemotactic protein 1; MSC, mesenchymal stem cell; SDF1, stromal cell derived

factor 1; TE/RM, tissue-engineered/regenerative medicine; TNFR1, tumor necrosis factor receptor 1; VEGF, vascular endothelial growth factor.

bSee the presentations to the Cellular, Tissue and Gene Therapies Advisory Committee Meeting, November 17, 2011 (http://www.fda.gov/AdvisoryCommittees/

CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/CellularTissueandGeneTherapiesAdvisoryCommittee/ucm284476.htm).

Review Trends in Biotechnology September 2013, Vol. 31, No. 9

attenuate NFkB signaling pathways: implications for aparacrine mechanism of immune regulation and regener-ative outcomes’ (Poster) (www.tengion.com/pdfs/Ilagan-2010-TERMIS-poster-FINAL.pdf)) (Box 3).

Development of reference materials, dose-response

curves, standards and controls

As with any assay, the development of robust positive andnegative controls is critical. A key requirement for anypotency assay is the ability to fail subpotent product ormaterials unrelated to the product. Negative controls forpotency assays may be developed through the intentionaldegradation of product by methods such as ageing, treat-ment with toxins, the application of environmental stress-ors, or mechanical damage. The reference standard ismaterial of the same type as the product that providesthe comparator for the potency assay, permitting the defi-nition of a potency ratio as the bioactivity of the product

512

sample per unit viable cell relative to the reference stan-dard. Reference standards may include well-characterizedclinical lots or preclinical study materials, or establishedcell lines that recapitulate bioactivity of the product can-didate.

Well-established reference standards may not be avail-able for product candidates, particularly for those at thepreclinical phase of development. Under these circum-stances, a reference standard may be developed over timeas progressively more data is generated from multipleclinical samples. These data may then be averaged tocreate an overall reference standard. Obviously, this strat-egy will be minimally useful during initial development ofthe potency assay but will become progressively morerelevant over time. Alternatively, a reference standardmay be designated arbitrarily for the purposes of thebioassay, with all outcomes normalized relative to thisreference standard [37].

Box 4. Outstanding questions

What is the specific mechanism of action for each product candidate?

Definition of the mechanism of action/mechanisms of action (MOA/

MOAs) of the product is a prerequisite for the development of a

functionally relevant bioassay. To this end, clear and rigorous

elucidation of how cell or cell–biomaterial-based product candidates

mediate regenerative outcomes remains forthcoming. Currently

fashionable, particularly for mesenchymal stem cell like cell therapies,

are references to paracrine-mediated action-at-a-distance effects

catalyzed through well-established regenerative factors, such as

vascular endothelial growth factor or hepatocyte growth factor [11–

13,17]. However, in the absence of a definitive in vivo functional

evaluation of a product that is genetically modified to be a null

expresser for such cytokines, these assays may simply be an artifact of

investigator bias and have little relevance to actual product MOA/

MOAs.

How can product MOA/MOAs be established?

The resolution of the MOA/MOAs of the product requires an under-

standing of the biodistribution of the product in vivo over time. What

is the fate of the cellular and biomaterial components of the product

candidate? What signaling and secretory elements of the cellular

component are functionally relevant? These questions can only be

addressed rigorously by labeling the product candidate through

genetic or molecular methods and following its biodistribution in

vivo. Similarly, the determination of functionally significant pathways

requires genetic knockout within the product of each candidate

signaling element followed by in vivo bio-analysis in a disease model.

Notwithstanding the fact that such experiments are technically

challenging, time-consuming and expensive (all factors that are

viewed negatively within industry), they lead directly to the next

unresolved question.

Is a labeled or otherwise modified product candidate functionally

equivalent to the original?

Standard operating procedure within academic laboratories for

elucidation of the MOA/MOAs of cell based therapies inevitably

involves labeling of the cellular component with trackable dyes or

magnetic particles, or through genetic modification with reporter

constructs. However, tissue-engineered/regenerative medicine pro-

ducts or product candidates have typically been through extended in

vivo bio-safety and proof-of-concept studies before selection for

evaluation in clinical trials and manufacturing. Within this context,

any modification to the product, no matter how trivial, raises concerns

regarding the equivalence between the modified product and the

original. Has the modification to the product that is required for

understanding the MOA/MOAs functionally altered the product such

that it may no longer be regarded as the same product? How would

this even be demonstrated? The default position currently adopted by

the industry is to assume that any modification, no matter how trivial,

represents a fundamentally new product and is therefore not

acceptable as it requires all previous in vivo bio-safety and functional

studies to be repeated.

This position can only be altered by detailed and rigorous functional

studies using transcriptomic, proteomic, secretomic, and related

methodologies to demonstrate functional equivalence between product

candidates with and without labeling and/or genetic modification.

Review Trends in Biotechnology September 2013, Vol. 31, No. 9

Concluding remarksThe establishment of a consistent, global framework forpotency assay development relevant to the TE/RM indus-try remains forthcoming. So far, the definition and estab-lishment of potency assays has been made on a product-by-product basis by the sponsoring companies, which havebeen left to individually interpret general guidelines fromthe FDA. A substantial difficulty remains the ambiguity inproduct MOA/MOAs and how this may be resolved (Box 4).The relatively small number of cell based therapies cur-rently in or about to enter clinical trials has led to a lack ofclear guiding precedents, especially for tissue-engineeredneo-organs and neo-tissues. To this end, we haveattempted to define a minimal framework of guiding prin-ciples for the TE/RM industry based on the few establishedproducts and product candidates currently under develop-ment (Table 2):(i) The potency of TE/RM products is defined as:

(Functional bioactivity of product candidate/unitviable cell)/(Functional bioactivity of reference stan-dard/unit viable cell).

(ii) Cell number and cell viability are not sufficient asindices of product potency but are required to expresspotency quantitatively as indicated above.

(iii) Potency assays should leverage the product MOA orother therapeutically relevant bioactivity.

(iv) An iterative, matrix-based, phase-specific potencyassay development and implementation regimen isrecommended for TE/RM products.

(v) The NUC and NKA product candidates serve asillustrative examples for the development andimplementation of potency assays for TE/RM pro-ducts. The NUC and NKA products leverage a battery

of functional bioassays linked to MOA, including cellmigration and secretion of growth factors andmicroRNAs.

As detailed in Box 4, future research will serve tospecifically identify critical MOA/MOAs pertinent to indi-vidual TE/RM product candidates. Understanding theMOA/MOAs on a product-by-product basis representsthe gateway to the development of functionally relevantpotency assays that will allow the pipeline of nascent TE/RM product candidates to navigate the regulatory frame-work of the FDA with minimal difficulty.

Disclaimer statementThe authors declare an equity and intellectual propertyinterest in Tengion, Inc.

AcknowledgmentsWe thank Randal McKenzie (McKenzie Illustrations, rmac3@att.net) forthe graphics in Figure I Box 1, Figure I Box 2, and Figure I Box 3.

References1 Basu, J. and Ludlow, J.W. (2012) Developmental engineering the

kidney: leveraging principles of morphogenesis for renalregeneration. Birth Defects Res. C: Embryo Today 96, 30–38

2 Andree, B. et al. (2013) Small intestinal sub-mucosa segments asmatrix for tissue engineering: review. Tissue Eng. Part B: Rev. 19,279–291

3 Carmen, J. et al. (2012) Developing assays to address identity, potency,purity and safety: cell characterization in cell therapy processdevelopment. Regen. Med. 7, 85–100

4 Rayment, E.A. and Williams, D.J. (2010) Mind the gap: challenges incharacterizing and quantifying cell and tissue based therapies forclinical translation. Stem Cells 28, 996–1004

5 Basu, J. and Ludlow, J.W. (2010) Platform technologies for tubularorgan regeneration. Trends Biotechnol. 28, 526–533

513

Review Trends in Biotechnology September 2013, Vol. 31, No. 9

6 Basu, J. and Ludlow, J.W. (2011) Tissue engineering of tubular andsolid organs: an industry perspective. In Advances in RegenerativeMedicine (Wislet-Gendebien, S., ed.), pp. 235–260, Intech Open

7 Bravery, C.A. et al. (2013) Potency assay development for cellulartherapy products: an ISCT review of the requirements andexperiences in the industry. Cytotherapy 15, 9–19

8 Center for Biologics Evaluation and Research (2011) Guidance forIndustry: Potency Tests For Cellular And Gene Therapy Products.US Food and Drug Administration

9 Kretlow, J.D. et al. (2008) Donor age and cell passage affectsdifferentiation potential of murine bone-marrow derived stem cells.BMC Cell Biol. 9, 60

10 Prockop, D. (2009) Repair of tissues by adult stem/progenitor cells(MSCs): controversies, myths and changing paradigms. Mol. Ther. 17,939–946

11 Caplan, A.I. and Correa, D. (2011) The MSC: an injury drugstore. CellStem Cell 9, 11–15

12 Jiao, J. et al. (2011) A mesenchymal stem cell potency assay. MethodsMol. Biol. 677, 221–231

13 Bai, L. et al. (2012) Hepatocyte growth factor mediates mesenchymalstem cell-induced recovery in multiple sclerosis models. Nat. Neurosci.15, 862–870

14 Basu, J. et al. (2011) Functional evaluation of primary renal cell/biomaterial neo-kidney augment prototypes for renal tissueengineering. Cell Transplant. 20, 1771–1790

15 Xu, Q. et al. (2009) Constitutive release of cytokines by human oralkeratinocytes in an organotypic culture. J. Oral Maxillofac. Surg. 67,1256–1264

16 Camussi, G. et al. (2013) Role of stem cell derived micro-vesicles in theparacrine action of stem cells. Biochem. Soc. Trans. 41, 283–287

17 Lehman, N. et al. (2012) Development of a surrogate angiogenicpotency assay for clinical grade stem cell production. Cytotherapy14, 994–1004

18 Medicetty, S. et al. (2012) Percutaneous adventitial delivery ofallogeneic bone marrow-derived stem cells via infarct-related arteryimproves long-term ventricular function in acute myocardialinfarction. Cell Transplant. 21, 1109–1120

19 Mansbridge, J. (2006) Commercial considerations in tissueengineering. J. Anat. 209, 527–532

20 Basu, J. et al. (2011) Expansion of the human adipose derived stromalvascular cell fraction yields a population of smooth muscle-like cellswith markedly distinct phenotypic and functional properties relative tomesenchymal stem cells. Tissue Eng. Part C: Methods 17, 843–860

21 Basu, J. and Ludlow, J.W. (2012) Developments in Tissue Engineeredand Regenerative Medicine Products: A Practical Approach. WoodheadPublishing

22 Jayo, M.J. et al. (2008) Early cellular and stromal responses in theregeneration of a functional mammalian bladder. J. Urol. 180, 392–397

23 Basu, J. et al. (2012) Regeneration of native-like neo-urinary tissuefrom non-bladder cell sources. Tissue Eng. Part A 18, 1025–1034

24 Shah, S. et al. (2012) Cellular basis of tissue regeneration by omentum.PLoS ONE 7, e38368

514

25 Guthrie, K. et al. (2013) Migration assay to evaluate cellularinteractions with biomaterials for tissue engineering/regenerativemedicine applications. Methods Mol. Biol. 1001, 189–196

26 Basu, J. et al. (2012) Extension of bladder-based organ regenerationplatform for tissue engineering of esophagus. Med. Hypotheses 78,231–234

27 Basu, J. et al. (2011) Regeneration of rodent small intestine tissuefollowing implantation of scaffolds seeded with a novel source ofsmooth muscle cells. Regen. Med. 6, 721–731

28 Soncin, S. et al. (2009) A practical approach for the validation ofsterility, endotoxin and potency testing of bone marrowmononucleated cells used in cardiac regeneration in compliancewith good manufacturing practice. J. Transl. Med. 7, 78

29 Dahl, S. et al. (2011) Readily available tissue engineered vasculargrafts. Sci. Transl. Med. 3, 68ra9

30 Kelley, R. et al. (2013) A population of selected renal cells augmentsrenal function and extends survival in the ZSF1 model of progressivediabetic nephropathy. Cell Transplant. 22, 1023–1039

31 Guimaraes-Souza, N.K. et al. (2012) In vitro reconstitution of humankidney structures for renal cell therapy. Nephrol. Dial. Transplant. 27,3082–3090

32 Noiman, T. et al. (2011) A rapid in vivo assay system for analyzingthe organogenetic capacity of human kidney cells. Organogenesis 7,140–144

33 Genheimer, C. et al. (2012) Molecular characterization of theregenerative response induced by intra-renal transplantation ofselected renal cells in a rodent model of chronic kidney disease.Cells Tissues Organs 196, 374–384

34 Zeisberg, M. et al. (2002) Extra-cellular matrix microenvironmentregulates migratory behavior of activated tubular epithelial cells.Am. J. Pathol. 160, 2001–2008

35 Kelley, R. et al. (2010) A tubular cell-enriched subpopulation of primaryrenal cells improves survival and augments kidney function in a rodentmodel of chronic kidney disease. Am. J. Physiol. Renal Physiol. 299,F1026–F1039

36 Dudas, P.L. et al. (2009) BMP7 fails to attenuate TGFb1 inducedepithelial-mesenchymal transition in human proximal tubuleepithelial cells. Nephrol. Dial. Transplant. 24, 1406–1416

37 Hall, H. et al. (2012) Hematopoietic stem cell potency for cellulartherapeutic transplantation. In Advances in Hematopoietic StemCell Research. Intech Open

38 Roh, J.D. et al. (2010) Tissue-engineered vascular grafts transform intomature blood vessels via an inflammation mediated process of vascularremodeling. PNAS 107, 4669–4674

39 Ratliff, B.B. et al. (2010) Mesenchymal stem cells, used as bait, disclosetissue binding sites: a tool in the search for the niche? Am. J. Pathol.177, 873–883

40 Higano, C.S. et al. (2010) Sipulecel-T. Nat. Rev. Drug Discov. 9,513–514

41 Schwartz, S.D. et al. (2012) Embryonic stem cell trials for maculardegeneration: a preliminary report. Lancet 379, 713–720