Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications

Baghbaderani, Behnam Ahmadian; Adhikarla, Syama; Sivapatham, Renuka; Pei, Ying; Mukherjee, Odity; Fellner, Thomas; Zeng, Xianmin; Rao, Mahendra S.

doi:10.1007/s12015-016-9662-8

Cited by 63 publications

(57 citation statements)

References 56 publications

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“…More recently, cGMP‐compliant hiPSCs have been generated by teams in People's Republic of China , Japan , the U.S. and the U.K. . These clinically compliant lines have been extensively characterized as pluripotent with profiles comparable to previously validated hPSCs derived outside of these manufacturing guidelines . However, given previous reports describing the varied potential for hPSC lines to differentiate into target cell types , evaluation of their hepatic differentiation potential represents an essential prerequisite to further clinical development work.…”

Section: Introductionmentioning

confidence: 99%

Validation of Current Good Manufacturing Practice Compliant Human Pluripotent Stem Cell-Derived Hepatocytes for Cell-Based Therapy

Blackford

Segal

et al. 2018

Stem Cells Translational Medicine

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Recent advancements in the production of hepatocytes from human pluripotent stem cells (hPSC‐Heps) afford tremendous possibilities for treatment of patients with liver disease. Validated current good manufacturing practice (cGMP) lines are an essential prerequisite for such applications but have only recently been established. Whether such cGMP lines are capable of hepatic differentiation is not known. To address this knowledge gap, we examined the proficiency of three recently derived cGMP lines (two hiPSC and one hESC) to differentiate into hepatocytes and their suitability for therapy. hPSC‐Heps generated using a chemically defined four‐step hepatic differentiation protocol uniformly demonstrated highly reproducible phenotypes and functionality. Seeding into a 3D poly(ethylene glycol)‐diacrylate fabricated inverted colloid crystal scaffold converted these immature progenitors into more advanced hepatic tissue structures. Hepatic constructs could also be successfully encapsulated into the immune‐privileged material alginate and remained viable as well as functional upon transplantation into immune competent mice. This is the first report we are aware of demonstrating cGMP‐compliant hPSCs can generate cells with advanced hepatic function potentially suitable for future therapeutic applications. stem cells translational medicine 2019;8:124&14

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Section: Introductionmentioning

confidence: 99%

Validation of Current Good Manufacturing Practice Compliant Human Pluripotent Stem Cell-Derived Hepatocytes for Cell-Based Therapy

Blackford

Segal

et al. 2018

Stem Cells Translational Medicine

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“…Also beyond the scope of this article is a more detailed discussion of the many additional tests that hiPSCs should be subjected to following their derivation (see Baghbaderani et al., , for additional information) and how to validate and perform such tests in a compliant manner. At a minimum, before using an hiPSC line in downstream applications, it should be subjected to (ideally by a CLIA‐certified facility and/or using properly qualified, validated, executed and documents procedures): …”

Section: Commentarymentioning

confidence: 99%

Xeno‐Free Reprogramming of Peripheral Blood Mononuclear Erythroblasts on Laminin‐521

Skorik

Mullin

Shi

et al. 2020

CP Stem Cell Biology

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Translating human induced pluripotent stem cell (hiPSC)–derived cells and tissues into the clinic requires streamlined and reliable production of clinical‐grade hiPSCs. This article describes an entirely animal component–free procedure for the reliable derivation of stable hiPSC lines from donor peripheral blood mononuclear cells (PBMCs) using only autologous patient materials and xeno‐free reagents. PBMCs are isolated from a whole blood donation, from which a small amount of patient serum is also generated. The PBMCs are then expanded prior to reprogramming in an animal component–free erythroblast growth medium supplemented with autologous patient serum, thereby eliminating the need for animal serum. After expansion, the erythroblasts are reprogrammed using either cGMP‐grade Sendai viral particles (CytoTune™ 2.1 kit) or episomally replicating reprogramming plasmids (Epi5™ kit), both commercially available. Expansion of emerging hiPSCs on a recombinant cGMP‐grade human laminin substrate is compatible with a number of xeno‐free or chemically defined media (some available as cGMP‐grade reagents), such as E8, Nutristem, Stemfit, or mTeSR Plus. hiPSC lines derived using this method display expression of expected surface markers and transcription factors, loss of the reprogramming agent–derived nucleic acids, genetic stability, and the ability to robustly differentiate in vitro to multiple lineages. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Isolating peripheral blood mononuclear cells using CPT tubes Support Protocol 1: Removal of clotting factors to produce serum from autologous plasma collected in Basic Protocol 1 Basic Protocol 2: PBMC expansion in an animal‐free erythroblast expansion medium containing autologous serum Basic Protocol 3: Reprogramming of expanded PBMCs with Sendai viral reprogramming particles Alternate Protocol: Reprogramming of expanded PBMCs with episomal plasmids Basic Protocol 4: Picking, expanding, and cryopreserving hiPSC clones Support Protocol 2: Testing Sendai virus kit–reprogrammed hiPSC for absence of Sendai viral RNA Support Protocol 3: Testing Epi5 kit–reprogrammed hiPSC for absence of episomal plasmid DNA Support Protocol 4: Assessing the undifferentiated state of human pluripotent stem cell cultures by multi‐color immunofluorescent staining and confocal imaging Support Protocol 5: Coating plates with extracellular matrices to support hiPSC attachment and expansion

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“…The human LiPSC18R induced pluripotent stem cells (iPSC) line was generated from CD34+ cord blood cells as previously described [36]. RTiPSC3B and RTiPSC4i were generated from human peripheral blood mononuclear cells (PBMNCs, Lonza, CC-2702, Walkersville, MD, USA) of two different donors.…”

Section: Human Ipsc Linesmentioning

confidence: 99%

End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

Pandey

Tomney

Woon

et al. 2019

IJMS

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Industrialization of stem-cell based therapies requires innovative solutions to close the gap between research and commercialization. Scalable cell production platforms are needed to reliably deliver the cell quantities needed during the various stages of development and commercial supply. Human pluripotent stem cells (hPSCs) are a key source material for generating therapeutic cell types. We have developed a closed, automated and scalable stirred tank bioreactor platform, capable of sustaining high fold expansion of hPSCs. Such a platform could facilitate the in-process monitoring and integration of online monitoring systems, leading to significantly reduced labor requirements and contamination risk. hPSCs are expanded in a controlled bioreactor using perfused xeno-free media. Cell harvest and concentration are performed in closed steps. The hPSCs can be cryopreserved to generate a bank of cells, or further processed as needed. Cryopreserved cells can be thawed into a two-dimensional (2D) tissue culture platform or a three-dimensional (3D) bioreactor to initiate a new expansion phase, or be differentiated to the clinically relevant cell type. The expanded hPSCs express hPSC-specific markers, have a normal karyotype and the ability to differentiate to the cells of the three germ layers. This end-to-end platform allows a large scale expansion of high quality hPSCs that can support the required cell demand for various clinical indications.Int. J. Mol. Sci. 2020, 21, 89 2 of 29 recapitulate in vivo conditions. To replace the number of cells lost during a myocardial infarction, for example, approximately 1 × 10 9 cells are required per patient dose [7].Given that 2D-based cell culture platforms are nonscalable with minimal capacity for expansion, achieving high cell densities in a 2D system would involve costly arrangements including extensive manual effort, laboratory space and personnel. These platforms also often do not possess adequate systems to control or monitor parameters, such as the production of key metabolites by hiPSCs in culture. Moreover, iPSC-derived cardiomyocytes remain phenotypically immature [8], despite a number of studies demonstrating enhanced maturation through the modulation of existing methodologies [9][10][11][12][13].Recent innovations in suspension culture systems provide robust, controlled and scalable platforms beyond conventional 2D approaches, which can be translated to current Good Manufacturing Practice (cGMP) compliant processes [14][15][16]. A number of studies have demonstrated the feasibility of hPSC expansion in suspension cultures using aggregate [14,16,17] and microcarrier (MC)-based [18][19][20] three dimensional (3D) culture systems. Aggregate-based 3D culture provides a more physiologically relevant microenvironment, but has been shown not only to require the small molecule, Y27632, for the survival of hPSCs [15], but also sequential passaging to achieve high fold expansion [21]. Not without its own advantages, microcarrier-based culture systems facilitate a larger surface...

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Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications

Cited by 63 publications

References 56 publications

Validation of Current Good Manufacturing Practice Compliant Human Pluripotent Stem Cell-Derived Hepatocytes for Cell-Based Therapy

Validation of Current Good Manufacturing Practice Compliant Human Pluripotent Stem Cell-Derived Hepatocytes for Cell-Based Therapy

Xeno‐Free Reprogramming of Peripheral Blood Mononuclear Erythroblasts on Laminin‐521

End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

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