key: cord-0955502-v6wpnyfi
authors: Chakrabarty, Koushik; Shetty, Rohit; Argulwar, Shubham; Das, Debashish; Ghosh, Arkasubhra
title: iPSC-based disease modeling and prospective immune therapy for COVID-19
date: 2021-09-14
journal: Cytotherapy
DOI: 10.1016/j.jcyt.2021.08.003
sha: 808674e16292b72667d99392de41b4b448754dc3
doc_id: 955502
cord_uid: v6wpnyfi

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses a never before seen challenge to human health and economy. Considering its clinical impact with no streamlined therapeutic strategies in sight, it is crucial to understand the infection process of the SARS-CoV-2. Our nondescript knowledge of the foresaid mechanisms impedes development of alternative therapeutics to address the pandemic. This aspect can be addressed by modeling SARS-CoV-2 infection in the human context to facilitate drug screening and discovery. Human induced pluripotent stem cells (iPSCs) derived lung epithelial cells and organoids recapitulating the features and functionality of the alveolar cell types can serve as an in vitro human model and screening platform for SARS-CoV-2. Recent studies suggest an immune system asynchrony leading to compromised function and proportion of specific immune cell types in Coronavirus disease (COVID) -19 patients. Replenishing these specific immune cells may serve as useful effectors against SARS-CoV-2 infection. Here, we review protocols for deriving lung epithelial cells, alveolar organoids and specific immune cells types such as T- lymphocytes and natural killer (NK) cells from iPSCs with the aim to aid investigators to make relevant in vitro models of SARS-CoV-2 along with the possibility derive immune cells types to treat COVID-19.

-iPSC based COVID-19 disease model for novel drug discovery -iPSC derived immunocompetent cell therapy for COVID-19

Introduction SARS-CoV-2 have remarkably and very rapidly adapted to humans, their recent host. Most of the mechanistic knowledge about the host specific response and the virus-host interplay are extrapolated from the knowledge obtained from infection of other members of coronavirus family [1, 2] . Lack of extensive understanding in the underlying infectivity and response between the virus and novel host (human being) are hindering development of appropriate treatment strategies. With the continuing increase in SARS-CoV-2 infection and associated mortality across the globe, it is imperative to devise an in-vitro SARS-CoV-2 infection model for providing the possibility to unravel the strain virulence, disease process paving way to facilitate drug screening and develop novel treatment strategies for improving the clinical outcome. Currently immortalized cell lines are being utilized to model the permissiveness features of SARS-CoV-2 and also propagate them [3, 4] . However, these models lack the physiological relevance and as immortalized cell lines are prone to contain genomic aberrations limiting its scope as biologically relevant model of SARS-CoV-2 [5] .

Although these cell lines are effective in their capability to propagate SARS-CoV-2 they are inept in recapitulating the features and function of the human airway epithelium therefore emphasizing the need for a relevant and a contextual in vitro system to study SARS-CoV-2 kinetics, trophism and host response [6] . Studies on the trophism of the SARS-CoV-2 has demonstrated its specific affinity to the respiratory epithelium, which is also its primary route of entry due to high expression and activity of the angiotensin converting enzyme 2 (ACE2) receptor, transmembrane protease serine 2 (TMPRSS) and cathepsin B [7] . ACE2 and TMPRSS2 have been detected in both nasal and bronchial epithelium [8] . Gene expression of ACE2 and TMPRSS2 has been reported to occur largely in alveolar epithelial type II (AT2) cells [9] which are central to SARS-CoV-2 pathogenesis. Human iPSCs having the unique properties of unlimited self-renewal capacity and can potentially differentiate into various types of somatic cells [10] . iPSCs offers the possibilities to generate alveolar epithelial cells and 3-dimentional (3D) lung organoids as an in vitro predictive model for SARS-CoV-2 infection and as a screening platform for COVID-19 therapeutics [11, 12] . Such an iPSC derived model of respiratory/alveolar tissue would serve as a platform for large scale screening of anti-viral drugs apart from understanding the disease mechanism [13] . Current scenario with the large number of high-risk/ immune compromised COVID-19 patients around the globe necessitates pursuing multiple approaches simultaneously in order to achieve the best outcomes for the patients and society at large in terms of curative and preventive care. Clinical findings in COVID-19 patients attribute a dysregulated/exuberant immune response as a leading contributor to SARS-CoV-2 mediated pathology. Interestingly, the acute phase of SARS in humans is associated with a severe reduction in the number of T cells in the blood [14] . So far, only a limited number of studies have explored the role of the T cell-mediated adaptive immune response in COVID-19 pathogenesis. A recent study showed T cell counts are reduced significantly and those surviving are functionally exhausted in COVID-19 patients [15] . The study also suggests the persistent low counts of CD8+ and CD4+T cells in non-ICU COVID-19 patients would need aggressive intervention even in the immediate absence of severe symptoms to reduce risk for further deterioration in condition.

Based on these clinical insights development of 'off-the-shelf' third-party allogeneic virusspecific T cell therapies can be a powerful tool to treat COVID-19 associated lyphophenia, which is currently not amenable to standard therapeutic strategies. Like the T cells, the role of NK cells in SARS-CoV-2 infections is currently being examined [16] [17] [18] [19] [20] . The rationale of using NK cells as a modality for immune therapy for COVID-19 is also being considered.

Here, we discuss protocols to derive respiratory epithelial cells and alveolar organoids towards modeling the infection biology of the novel SARS-CoV-2 along with the possible applications of hiPSC derived T and NK cells as therapeutic intervention against this newly emerged pathogen.

Infectious viruses often are restricted to their specific hosts and cell types which are the primary routes of entry into the host system. Modeling this host-virus interaction is challenging, since most of the circulating human viruses are genetically different from viruses cultured under standard laboratory conditions. For example, the surface glycoproteins of the human parainfluenza virus 3 which is critical for its entry into the host cell differs considerably from the ones cultured on immortalized monolayer cells in the laboratory [21] The SARS-CoV-2 infection begins with its entry into the respiratory epithelial cells, which is mediated by the surface ACE2 receptor [7, 22] . The SARS-CoV-2 virus propagates within the infected epithelial cells including the AT2 (alveolar type II) cells, where in some cases the host innate immune response is triggered. Coincidently, most human pluripotent stem cells (PSCs) differentiation protocols has been directed to obtain AT2 positive cell phenotypes due to its crucial physiological role in vivo along with the possibility to evaluate its functionality by measuring expression and secretion of surfactant molecules [23, 24] . These findings underscore the need for recapitulating the infection process in an in vitro model where, the virus features are reasonably mimicked for infecting its choice of cell type.

Directed differentiation of iPSCs towards lung cells had presented with considerable initial challenges [25] . To address those challenges a rational stepwise approach mimicking the mammalian lung development in vivo has essentially been followed to successfully differentiate the iPSCs to functional alveolar cells [26, 27] . The key aim here is to obtain a pure population of lung epithelia with the ability to expand in vitro to perform disease modeling and harness it as a platform for novel drug development ( Figure 1 ). Defined protocols have now been established to derive specific lung lineages cell phenotypes (Table   1 ). Bonafide lung epithelial cells were obtained by Huang et al [28] by initially driving the pluripotent stem cells to a definitive endoderm fate, which were further directed to FOXA2 and NKX2.1 lung epithelial progenitor cell phenotype. The study also demonstrated FOXA2+NKX2.1+ double positive progenitor cells to give rise to basal, club, goblet, ciliated, alveolar type 1 (AT1) and alveolar type 2 (AT2) cells both in vivo and in vitro. Although yet to be established from human iPSCs, Garetta et al [29] using mouse embryonic stem (ES) and iPSC cells showed low oxygen tension significantly enhanced differentiation towards lung progenitor phenotype. Recently, Surendran et al [30] elucidated a protocol for generating lung epithelial cells from hiPSCs. Their protocol initially differentiated the iPSCS to a definitive endoderm fate, followed by progression into anteriorized endoderm which can give rise to both proximal and distal epithelial cells. Heo et al [27] derived alveolar epithelial cells from hiPSCs to evaluate plumotoxicity and inflammatory responses of toxins. In their protocol, the hiPSCs were differentiated for alveolar epithelial progenitor markers such as NKX2.1 (also known as thyroid transcription factor), epithelial cell adhesion molecule (EPCAM), and carboxypeptidase M (CPM). Robust differentiation to alveolar epithelial cells was obtained with more than 90% NKX2.1 positive cells, while the percentage of cells coexpressing NKX2.1 and EPCAM was approximately 70%. The differentiated cells also expressed pulmonary surfactant-associated protein B (SFTPB) and pulmonary surfactantassociated protein C (SFTPC) indicating typical functional signatures of AT2 cells. Since, AT2 have the potential to trans-differentiate into type 1 AT (AT1) cell type during normal lung development [31] or after lung injury [32] the authors determined the expression of the T1-alpha (T1A)/ podoplanin (cell membrane protein expressed specifically in AT1 cells) [33] to distinguish the fate of iPSC derived AT positive cells post 25 days of differentiation.

The authors also found comparable numbers of lung mesenchymal cells expressing PDGFRβ, CD90, NG2, and CD146 on day 25 of differentiation suggestive that the differentiation protocol also generates mesenchyme cell phenotypes found in the alveolar niches. Gotoh et al [34] differentiated human iPSCs to form NKX2-1+ "ventralized" anterior foregut endoderm cells, from which cells expressing carboxypeptidase M (CPM) were sorted for 3D coculture with fetal human lung fibroblasts. The resulting CPM+ organoids although devoid of club cells contained mostly AT2 cells, as well as some AT1, ciliated cells, and goblet cell. These However, a relatively homogeneous population of AT2 and AT1 cells was generated from human iPSCs reprogrammed from fetal or neonatal lung fibroblasts [36] . Human iPSCderived AT2 alveolospheres exhibited self-renewal capacity and displayed immune responsiveness [37] . These studies clearly indicate that alveolar cell lineages produced from iPSC-derived lung organoids mimics the development of their in vivo counterpart [38, 39] ( Figure 2 ). Incidentally, fibroblast growth factor (FGF) signaling has been demonstrated to be crucial to promote the induction of anterior foregut endoderm into human lung organoids encompassing the contextual cellular milieu [39] . Historically, polarized human airway epithelium (HAE) culture system has been used to represent authentic airway for respiratory virus infection [40, 41] . However, the HAE model represents few of the alveolar cell types which limit its utility as a cellular model in the scenario to study SARS-CoV-2. To address these issues modeling respiratory virus infection using lung organoids have been explored [42] [43] [44] as they encompass most of the pulmonary cell types and components present in their tissue counterpart more closely. Chen et al [43] reported derivation of 3D lung organoids from human pluripotent stem cells (hPSCs). These organoids recapitulated the developing 

iPSCs as a perpetual source of pluripotent stem cells can be directed to be differentiated towards the lymphoid lineage [46] . This aspect of the technology allows having a continuous production of lymphocytes thereby addressing the bottlenecks such as cell numbers and dose limitations seen with respect to primary lymphoid cells. Therefore, compared to primary lymphoid source, iPSCs can be used to develop a robust and safe platform to develop "off the shelf"" immune cell therapeutics to address COVID-19 ( Figure 1 ). So far, iPSCs has been generated from almost all types of somatic cells [47] . However selection of the initial iPSC source is critical for their latter derivation of lymphoid lineage cells. Reminiscent epigenetic memory of the cells is known to play a crucial role in deciding the differentiation fates of iPSCs [48, 49] . How epigenetic memory is retained and paves for preferential differentiation to a specific cellular lineages has been reviewed before [46, 48] and might explain why iPSCs generated from cells having hematopoietic lineage are more successfully differentiated towards lymphoid lineages.

Incidentally, SARS in humans is associated with a severe reduction in the number of T cells in the blood similar to that observed in COVID-19 patients [50] . In addition, Zheng et al reported elevated exhaustion levels and reduced functional diversity of T cells in peripheral blood in COVID-19 patients predictive of disease severity [51] . The reason for T cell exhaustion in COVID-19 patients can be attributed to their repeated activation which also reduces the possibility to harvest these cells from the COVID-19 patients for adoptive cytotoxic T lymphocyte (CTL) therapy. CTLs play an important role in the immune-system defense against viral infections. CTLs interact with the virus infected cells triggering a cascade which eventually kills the infected cells [52] . The key player in this cascade are the major histocompatibility complex (MHC) class I molecules which presents the antigenic peptides activating the CTLs leading to their degranulation and subsequent death of the target cells [53] . This crucial mechanism which protects us from virulent pathogens takes effect by virtue of a very specific phenomenon called "MHC restriction" where the MHC allele is recognized by the patient"s own T cells only. Therefore "MHC restriction" is considered to be the primary constraint for adoptive T cell therapy [54] . More so towards employing an adoptive T cell therapy in the context to treat COVID 19 where the quality of the patient derived primary CTLs could be not optimum. This aspect can undermine its expansion potential to obtain enough CTL for treating a large number of COVID-19 patients. Human iPSCs, with its unique features of pluripotency and infinite propagation can address this critical constraint by serving as an alternative source to derive T cells for allogenic ""off the shelf"" adoptive immunotherapy [55] to treat COVID-19 patients with lymphopenia ( Figure   1 ). Some crucial insight in this direction has been obtained from the work of Nagano et al [56] who successfully derived potent tumor antigen specific CTLs from T-iPSCs in a allogenic setting. More importantly, they also underline the issues that need to be addressed prior to the allogenic application of the CTLs in the clinic. With the above finding, it can be conjured that the ability to derive universal donor T cells from gene modified PSC lines [57] can lead to the development of a allogeneic virus-specific T cell therapies to treat COVID-19 associated lymphopenia which are currently not amenable to standard therapeutic strategies.

Derivation of immunocompetent and therapeutic T lymphopoiesis from pluripotent stem cells (PSCs) can replenish the T cell reduction seen in severe COVID-19 cases [15] . To date, generation of fully functional and mature T cell from PSCs remains a challenge. Addressing the challenges, Montel-Hagen et al [58] reported that a 3D artificial thymic organoid (PSC-ATO) culture system which allows in vitro differentiation of hiPSCs to functional and mature T cells. The authors here harnessed the Notch-ligand expressing stromal cells line in a serum free condition [59] to obtain CD4+CD8+ double positive precursors towards directed differentiation of functional T cells. Interestingly, T cells reprogrammed to induced pluripotent stem (iPS) cells and were latter driven to differentiate to T lymphocyte (T-iPS cells) are found to preserve the intricate and precise re-arrangements of the α and β genes of the T cell receptor (TCR) identical to the original T cell clone [60] . Furthermore, theses redifferentiated CD8 single positive T cells from the T-iPSCs displayed increased telomere length suggestive of their rejuvenated status. Alongside, these T-iPSCs derived T lymphocytes also exhibited antigen-specific cytotoxicity and increased proliferative response which can be attributed to their rejuvenation. The approach here was based on reprogramming CD8+ T cells first into iPSC (to retain the epigenetic memory of the parental cell [61] ) which were called T-iPSC based on the cell type source and their subsequent stepwise directed differentiation to hematopoietic lineage, T cell progenitors and finally deriving functionally matured CD8+ T cells ( Figure 2 ). These protocols provide useful in vitro tools to generate antigen-specific T cell for immunotherapy ( Table 2 ).

The key to recognize antigens by T cells is by virtue of the TCRs, encoded by uniquely rearranged genomic loci of the TCR alpha and ß chains [62] . Generally, deriving T cells from iPSCs lines has been shown to bear non-rearranged germline TCR loci, resulting in random rearrangement during differentiation. Nishimura et al [62] also confirmed that specificity of TCR rearrangement remains unchanged in T cells derived from T-iPSCs. These two studies provide the crucial evidence to the possibility to derive a therapeutic anti SARS-CoV-2specfic T cells from T-iPSC generated from COVID-19 patients. Demonstrating safety of iPSC derived T cells, Ando et al [63] reported enhancing the safety of iPSC derived rejuvenated T cells by introducing inducible caspase-9 (iC9). The study demonstrated that activation of iC9 eliminated the iPSCs derived rejuvenated cytotoxic T lymphocytes without disturbing their antigen-specific killing activity suggesting the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.

Natural Killer (NK) cells are lymphoid cells that originate from the same progenitor as T cells and B cells. They are an important part of the innate immune response where they identify the "non-self" cells independent to antigen presentation or recognition thereby executing a rapid immune response. The broad cytotoxicity and rapid apoptosis induced by the NK cells help contain virus-infected cells while the adaptive immune response is activated to produce cytotoxic T cells to clear the antigens. So far primary NK cells have been the main source used for immunotherapy. Significant work is being done to obtain NK cells from other sources such as iPSC to have a perpetual source of NK cells for therapy.

Primary NK cells are difficult to harvest and purify [64] . They are also difficult to standardize due to the heterogeneity in starting material from different donors [65] . As well, the generation of large quantities of highly pure NK cells requires an extended manufacturing process which can compromise recovery of NK cells, its viability and potency [66] . NK cells derived from iPSCs have proven to be equally as effective as primary NK cells [67, 68] . The perspective of generating NK cells from iPSCs may address some of the concerns related to adoptive NK cell therapy. Studies have demonstrated the possibility to derive NK cells robustly with its characteristic phenotypic features from iPSCs [69] [70] [71] . Most importantly, Knorr et al, [72] reported a clinical-grade, serum-free, and feeder-free differentiation protocol to obtain functional NK cells from iPSC, which involved a novel technique for generating the intermediate hematopoietic embryoid bodies using defined xenofree conditions and membrane bound interleukin 21-expressing artificial antigen-presenting cells. According to the authors, this method allows to obtain sufficient number of functional cytotoxic NK cells derived from as little as 250,000 PSCs to treat a single patient thus facilitating the potential for its clinical application. NK cell based COVID-19 treatment is currently being considered [73] . However, currently there is little evidence of the iPSC-NK cells requiring the same activation pathways for achieving its functionally attributes. Further studies are needed to understand the activation process of the iPSC derived NK cells for generating functionally efficient iPSC-NK cells. With our current understanding , these data suggest that iPSC-NK-based strategies (Table 3 ) combine the most attractive qualities of primary NK cells (such as high potential for cytotoxicity, including ADCC function, and potential for expansion and persistence in vivo after cryopreservation) making it an tempting immune cell candidate for COVID-19 therapy.

The challenges of using iPSC derived cells for modelling and as an immune therapy strategy to address covid-19 are intrinsic to the very biology of the iPSCs i.e. donor age, somatic cell type used for reprogramming and to what fate are they going to be differentiated [74] .

Retention of epigenetic memory of their parental somatic source by the induced pluripotent cells has been well documented feature of the reprogramming process [75] . This is considered to be one of the primary reasons for their clonal heterogeneity deciding their potential differentiation fates [76] . Although, longer passages of iPSCs have shown diluting the epigenetic memory it also increases the risk for incorporating random gene mutations leading to genomic instability [77] of the lines which is detrimental towards deriving relevant cell types for modelling and therapy. In the past decade rapid advances are being made to address the concerns relevant to iPSC application [78] . Knowledge gained here has led to formulating criteria"s for defining good iPSCs lines and their differentiated derivatives.

However, there is the need and sufficient space to rethink existing strategies and bring forth improved and robust acceptable standards for clinical application of iPSCs and their derivatives [79] . The development of practical, reliable and cost effective methods to obtain iPSC derived lungs cells, T cells and NK cells is a vital pre-requisite for its application for modelling and as a therapeutic prospect for addressing the COVID-19 pandemic. One of the major concerns of iPSC based immune cell replacement therapy is risk of potential tumorigenicity from the calcitrant iPSCs which has not undergone complete differentiation to the desired phenotype [80] . Here, any reminiscence of stemness in even a single cell amongst the differentiated population poses an absolute risk for cellular transplantation. With the continuous advancement in directed differentiation and purification protocols, such risks are being addressed [81, 82] . However, translational work in animal models is necessary to assess if these assays and methods are efficient to address the risk of teratomas upon transplantation of the iPSC derived immune cells in COVID-19 patients. iPSC derived alveolar cells and lymphoid cells can be immunogenic which remains a major issue for its therapeutic application [83, 84] . To alleviate, long term immunosuppression has been shown to be indispensable and prevents rejection of allogenic pluripotent stem cells derived products in animal studies [85] . This could be the probably route of intervention in humans receiving therapeutic allogenic iPSC derived cells [86] . On the other hand, in case of autologous iPSC derived product it is feasible to transplant the therapeutic cells without immune suppression [87] . Cost of developing iPSC derived alveolar and lymphoid cells are significant which can be addressed to some extent with allogenic iPSC derived immune cells to treat some cases of COVID-19. However, the use of immunosuppression furthers the risk to the already disadvantaged patients to severe secondary infections [88] . In this scenario the risk versus benefit ratio is highly debatable which is akin to treating any disease with allogenic pluripotent stem cells derived cell product. In context to the current scenario and urgency, a universal immune tolerant iPSC line derived product will provide the necessary advantage to devise strategies to enable utilization of the allogenic iPSC lines derived alveolar and lymphoid cells without the risk of immune rejection and other complications.

As of now, vaccines against SARS-CoV-2 has become the most viable global option with several COVID-19 vaccines currently in trial and use [89] . Human cell lines have been routinely used to make vaccines against viruses [90, 91] . Vaccines against viral diseases have been continuously evolving to provide a safer means of protection and reduce possible side effects [92] . The choice of cell substrate is one of the most crucial components in viral vaccine manufacturing. Vaccines against chickenpox are being made using MRC5, a human fibroblast cell line [91] . The human embryonic kidney (HEK)-293, a cell line generated from aborted foetus in the 1970s is the cell substrate of choice for many COVID-19 vaccines manufactures [93, 94] . The immortalized HEK cells are harnessed for making the spike proteins of SARS-COV-2 [95] or for cultivating the recombinant packaging viruses needed towards vaccine manufacturing [96] . Although safety, pharmacology, toxicity and potency of using HEK-293 as a platform for vaccine manufacture is well established [97] . HEK-293 being an immortalized cell line of cancerous origin runs the inherent risk of chromosomal and genetic aberrations due to the countless number of passages they would have undergone.

Similar to immortalized cell lines, iPSCs under prolonged periods of culture have been shown to be capable to accumulate chromosomal and genetic aberrations [98] . However, this risk can be mitigated by a large degree using differentiated lung or alveolar type cells derived from a well characterized iPSC lines. iPSCs derived from chicken have been used in studying viral infection and replication of avian viral diseases [99] . The use of such galline iPSCs (giPSCs) to make viruses replication-incompetent from their highly pathogenic form [100] adds to the immense application potential of such animal specific iPSCs for vaccine production over chicken eggs and embryos which are more prone to contamination risk and can also aid in cutting down cost of vaccine production. In the current scenario, the iPSC derived alveolar type cells exposed to SARS-CoV-2 virus would serve as an invaluable tool able to reproduce the genotypic and phenotypic aspects more closely associated with the infection [101] . Exosomes derived from iPSCs and their derivatives are being considered for packaging the messenger RNAs (mRNAs) encoding the SARS-CoV-2 antigen proteins [102] .

The functionality of such exosome packaged mRNAs will address some of the critical issues of vaccine scalability and stability faced by mRNA based vaccines [103] . Apart or their utility as "packaging factory" in vaccine manufacturing, the iPSC derived alveolar cells can significantly aid towards novel target identification and repurpose known and novel drug combinations against SARS-CoV-2 and lay the prospects for an enhanced preparedness for future viral pandemic.

This review intends to aid researchers to streamline and optimize protocols to generate hiPSC derived alveolar cells and lung organoids as a robust platform to study respiratory viruses Author"s contributions: KC conceived and designed the paper. All authors contributed to the writing. KC took care of the editing, formatting, and submission. All authors read and approved the final manuscript Ethics approval: Not applicable Consent for publication: Not applicable Data availability statement: Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Competing interests: The authors declare that they have no competing interests. Figure 2 were created with BioRender software, ©biorender.com. Medium changed to anterior definitive endoderm (ADE) medium composed of DMEM + N2+B27+FBS +L-glutamine + Penicillin/streptomycin+ Noggin + SB431542 etc. D7-D12: Medium was changed to lung progenitor induction medium composed of ADE media without Noggin and SB431452. Wnt3A + hFGF10 + mFGF7 +BMP4+ hEGF +FGF2 and heparin sodium salt added for 5 days. D13-D23: Immature lung progenitor cells were grown into same medium for further 10 days to convert into mature cells. 

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D46-D60:The T lineage cells was harvested and mixed with irradiated HLA-A24_ PBMCs, and co-cultured using RH10 medium in the presence of IL-7 and IL- 15 doublepositive (DP) cells; once DP cells are generated, CD8ab single-positive (SP) cells was derived by stimulating the isolated DP cells.